Translation through ribosomes, amazing nano machines

Ribosomes amazing nano machines

https://reasonandscience.catsboard.com/t1661-translation-through-ribosomes-amazing-nano-machines

The ribosome in a ‘nutshell’
Internal Ribosome Entry Sites Provide Opportunities for Translational Control
The Ribosome is like a 3D printer
The ribosome as a missing link in the evolution of life
Assembly of the bacterial ribosome
Ribosome Biogenesis: An Overview
p53 and ribosome biogenesis stress: The essentials
Nervous-Like Circuits in the Ribosome
90S pre-ribosome transformation into the primordial 40S subunit
Quantum mechanic glimpse into peptide bond formation within the ribosome shed light on origin of life
Ribosome of escheria coli:
16S ribosomal RNA
Protein folding, surprising mechanisms point to an arranged set up
Amazing surveillance pathways that rescue ribosomes lost in translation point to intelligently designed mechanisms
The Ribosome is irreducibly complex
Structures of the human and Drosophila 80S ribosome
Syllogisms about the Ribosome

Ribosomes originated in the RNA world and increased in size over time. At the time of LUCA, the ribosome had largely formed Link

Lasse Lindahl (2022): Increasing Complexity of Ribosomes and Their Biogenesis
According to the classic ribosome model, developed in the 1960s and 1970s, its only function is to translate the four-letter nucleic acid code into the 20 amino acid peptide-code, while polymerizing amino acids into peptides with the help of a large complement of tRNAs and translation factors that cycle on and off the ribosome. However, advances accumulating over the recent decades have shown that the ribosome performs tasks beyond the classic model, such as initial folding of nascent peptides and regulation of translation in response to growth and stress conditions. Moreover, the ribosome interacts with the Signal Recognition Particle to secure the post-translation transport of protein products to their proper cellular location.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9332792/

The Nucleolus a Ribosome producing factory
https://reasonandscience.catsboard.com/t3039-the-nucleolus-a-ribosome-producing-factory

Error detection and repair during the biogenesis & maturation of the ribosome, tRNA's, Aminoacyl-tRNA synthetases, and translation: by chance, or design?
https://reasonandscience.catsboard.com/t2984-error-check-and-repair-during-messengerrna-translation-in-the-ribosome-by-chance-or-design

There are millions of protein factories in every cell. Surprise, they're not all the same 21 JUN 2017
A mammalian cell may harbor as many as 10 million ribosomes, and it can devote up to 60% of its energy to constructing them from RNA and 80 different types of proteins. Although ribosomes are costly, they are essential for translating the genetic code, carried in messenger RNA (mRNA) molecules, into all the proteins the cell needs. "Life evolved around the ribosome," Dinman says.
https://www.science.org/content/article/there-are-millions-protein-factories-every-cell-surprise-they-re-not-all-same

David L. Abel (2014): The translation process goes beyond just the mechanistic interactions between the polypeptide and ribosome tunnel. Internal mechanisms involving mRNA interactions occur by extension. Chaperone function occurs as an external mechanism. These mechanisms all work to contribute coherently to the folding process. The crucial point is that they are all dependent upon momentary pauses in the translation process. We collectively define these linked phenomena and their rate regulation as “co-translational pausing.” The dependency of folding on these multiple translation processes has been defined as “co-translational folding”. They reveal the ribosome, among other things, to be not only a machine but an independent computer-mediated manufacturing system.

T. Mukai et.al (2018) :Accurate protein biosynthesis is an immensely complex process involving more than 100 discrete components that must come together to translate proteins with high speed, efficiency, and fidelity. The E. coli ribosome alone is composed of54 proteins and 3 RNAs, whereas other translation factors include 33 tRNAs, 21 aminoacyl-tRNA synthetases, 3 initiation factors, 3 elongation factors, 2 release factors, and 12 nucleotide-modifying enzymes.

Conversations with Dimiter Kunnev about the Ribosome

https://www.youtube.com/watch?v=2guXr5c4rHc
Ribosomes are ancient and they've been around since life began.  Life began when ribosomes appeared




J.Monod: Chance and Necessity: An Essay on the Natural Philosophy of Modern Biology  12 setember 1972
The highly mechanical and even "technological" aspect of the translation process merits attention. The successive interactions of the various components intervening at each stage, leading to the assembly, residue by residue,  of a polypeptide upon the surface of the ribosome, like a milling machine which notch by notch moves a piece of work through to completion -all this inevitably recalls an assembly line in a machine factory.

Nowadays, it is a consensus that the ribosome should be understood as a prebiotic machine that predated the origin of cells
https://pubmed.ncbi.nlm.nih.gov/32764248/

Marco V. José (2020) Protein synthesis is the outcome of a complex translation system that involves ribozymes, ribosomal proteins, aminoacyl-tRNA synthetases (aaRSs), elongation and termination factors, and three kinds of RNA molecules, to wit, messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA)

Addy Pross:  How Was Nature Able to Discover Its Own Laws-Twice? 2021 Jul 12
Consider the capabilities of the ribosome, that microscopic entity located in the thousands in every living cell, and able to synthesize proteins from a supply of amino acids in an assembly-line type process, based on information coded into the cell’s DNA sequence. That molecular machine is able to churn out required proteins in the space of a few seconds [7]. The synthetic chemist, while able to combine amino acids to form peptides and simple proteins, can only gaze in wonder at the staggering efficiency and specificity of that ribosomal system.
https://pubmed.ncbi.nlm.nih.gov/34357051/

Jessica C. Bowman: The Ribosome Challenge to the RNA World 20 February 2015
The ribosome was fully functional at LUCA, forming a ‘‘common core’’. The common core rRNA, reasonably approximated by the rRNA of E. coli, is conserved over the entire phylogenetic tree. It contains universally conserved molecular structures. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.
https://williams.chemistry.gatech.edu/publications/LDW_105.pdf

Meredith Root-Bernstein The ribosome as a missing link in the evolution of life 21 February 2015
Hypothesize that ribosome was self-replicating intermediate between compositional or RNA-world and cellular life. We suggest that the ribosome may represent one important missing link between compositional (or metabolism-first), RNA-world (or genes-first) and cellular (last universal common ancestor) approaches to the evolution of cells.
https://pubmed.ncbi.nlm.nih.gov/25500179/

Mitch Leslie Origin and Evolution of the Ribosome 2010 Sep;
The modern ribosome was largely formed at the time of the last common ancestor, LUCA. Hence its earliest origins likely lie in the RNA world. The PTC and tRNAs clearly existed before LUCA.
https://www.sciencemag.org/news/2017/06/there-are-millions-protein-factories-every-cell-surprise-they-re-not-all-same

Bernard M.A.G. PietteA Peptide–Nucleic Acid Replicator Origin for Life March 11, 2020
Life as we understand it is cellular. The last universal common ancestor (LUCA) of all cells (not a single cell of course but a population) is understood in some detail; it possessed a cell membrane, DNA, the basic molecular machines for copying DNA (i.e., polymerase etc.), and a functional ribosome, among many more
https://www.cell.com/trends/ecology-evolution/fulltext/S0169-5347(20)30003-3#%20

Peptide bonds are synthesized at a rate of about 15 amino acids per second. Since three nucleotides constitute a codon for an amino acid, this means that the ribosome "moves" along the messenger RNA at the rate of about 3 x 15 = 45 nucleotides per second; that is the rate of translation is just as fast as the rate of transcription.

Translation is irreducibly complex
Translation needs mRNA with a initiation site, translation machinery, and termination site in mRNA. If one is missing, the genetic information cannot be translated into functional proteins.

Translation through the ribosome requires the coordinated action of at least three components:
1. The mRNA needs to have a translation initiation site, which includes a starting codon and surrounding sequences to establish the correct reading frame and the protein-coding region. Each mRNA has three potential reading frames. Consequently, the same mRNA could code for different proteins or no protein, depending on the context.
2. The host cell must have translation machinery that can recognize the starting codon.
3. A correct translation termination site must be recognizable by the translation termination molecules to release the mRNA and the translated protein product from the ribosome.

Complex artifacts made by man for specific purposes almost always require a manufacturing and assembly process which is more complex than the device to be made itself. I don't know of ANY factory, that makes products, that are equally complex, or more complex, than the factory itself, and the efforts to produce it. If we quantify the information, energy, and physical parts (machines, etc), and compare it to the product made, the former is always more complex than the latter. But remarkably, in life, in a VERY dramatic way, the opposite is the case. One single fertilized human egg stores the information, to make an organism, which, when grown up, is made of 37 trillion cells!! Science is not even close to unraveling how this is possible. And while human factories require a lot of human intervention, cell factories operate 100% autonomously.  Self-replication is the epitome of manufacturing sophistication. The machine at the core of the process in biology is the Ribosome. it requires several hundreds of assembly machines, which make the machines, that make the subunits of the Ribosome. Once each subunit is made, it goes through a very delicate, precise, and orchestrated test drive process. Even long-range communication between the assembly machines monitor if the newly synthesized ribosome subunit was produced properly, and only if the test drive is successful, the subunit is incorporated in the maturation of the ribosome. If not, there are proteasome grinders waiting to recycle the misfolded product, which, otherwise, would accumulate, and toxic the cell. Once the assembly factors have done their job, they are re-used in the next round to make the next ribosome. All this had to emerge prior to when life started, and so evolution was not the hero on the block. So one has either to believe, that all this enormously complex machine-building emerged spontaneously for no reason at all, or there was a super-intellect, that conceptualized life, and instantiated it, through his far superior intellectual capacity, than we humans have. Either chance or design. What is the superior, more rational explanation?

A. G. CAIRNS-SMITH Seven clues to the origin of life, 1990 page 48:
Now it is quite clear that the universality of all this higher-order organisation cannot be accounted for in terms of the pre-existence of precisely this organisation on a lifeless Earth. I don't think that anyone has suggested that the ribosome was picked out of a 'probiotic soup'.
https://3lib.net/book/808139/28f68a 

Life: What A Concept!
https://jsomers.net/life.pdf

Casey Luskin Leading Biologists Marvel at the “Irreducible Complexity” of the Ribosome, but Prefer Evolution-of-the-Gaps  Casey Luskin

George Church is Professor of Genetics at Harvard Medical School and Director of the Center for Computational Genetics
CHURCH: The ribosome, both looking at the past and at the future, is a very significant structure — it's the most complicated thing that is present in all organisms. Craig does comparative genomics, and you find that almost the only thing that's in common across all organisms is the ribosome. And it's recognizable; it's highly conserved. So the question is, how did that thing come to be? And if I were to be an intelligent design defender, that's what I would focus on; how did the ribosome come to be? Because it does a really great thing: it does this mutual information trick, but not from changing something kind of trivial, from DNA to RNA; that's really easy. It can change from DNA three nucleotides into one amino acid. That's really marvelous.
But isn't it the case that, if we take all the life forms we have so far, isn't the minimum for the ribosome about 53 proteins and 3 polynucleotides? And hasn't that kind of already reached a plateau where adding more genomes doesn't reduce that number of proteins?
VENTER: Below ribosomes, yes: you certainly can't get below that.
CHURCH: But that's what we need to do — otherwise they'll call it irreducible complexity. If you say you can't get below a ribosome, we're in trouble, right? We have to find a ribosome that can do its trick with less than 53 proteins.
VENTER: In the RNA world, you didn't need ribosomes.
CHURCH: But we need to construct that. Nobody has constructed a ribosome that works well without proteins.
VENTER: Yes.  To me the key thing about Darwinian evolution is selection. Biology is a hundred percent dependent on selection. No matter what we do in synthetic biology, synthetic genomes, we're doing selection. It's just not
natural selection any more. It's intelligently designed selection, so it's a unique subset. But selection is always part of it. 
VENTER: We have synthetic ribosomes in our lab, they're just not totally efficient right now. We didn't design them; we're copying the design.
https://evolutionnews.org/2008/02/leading_biologists_marvel_at_t/

Ludwig Maximilian University Biochemist studies how ribosomes make proteins 10 January 2017
Ribosomes are molecular machines programmed by genetic blueprints, which make proteins by linking amino acids together into linear chains that fold into sequence-dependent shapes. Protein production in cells is mass production. A single yeast cell may contain up to 200,000 ribosomes, a human liver cell may have up to a million. When one considers that an adult human is made up of over a billion cells, the magnitude of the task of the protein-synthesizing machinery, and its indispensability at every second of our existence, begins to dawn on us. Biological systems contain complex  dense networks of intermolecular communications that keep cells alive, each one representing a metastable system held together by sensors, signals and interactions.
https://phys.org/news/2017-01-biochemist-ribosomes-proteins.html

1. The set up of a language, and upon it, the programming of a completely autonomous communication network, which directs the operation of a complex factory, which during operation error checks and performs repairs, to make specific purposeful products, is always the product of an intelligent agency.
2. Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. They form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, they can halt the translation process on the fly, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.
3. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder, does science confess that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: an intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

Ribosomes must also perform many functions. These include:
(1) enhancing the accuracy of codon-anticodon pairing between the mRNA transcript and the aminoacyl-tRNAs,
(2) polymerizing (via peptidyl transferase) the growing peptide chain,
(3) acting as energy transducers converting chemical energy into the mechanical energy during translocation of amino acids from tRNA carriers,
(4) protecting the growing protein from attack by proteases (protein-degrading enzymes) possibly by forming a long protective tunnel, and
(5) assisting in the hydrolysis (dissolution) of the amino acid–tRNA bond during termination.

Further, several separate protein factors and cofactors facilitate various specialized chemical transformations during the three discrete steps of translation: initiation, elongation, and termination. In eukaryotes, initiating translation alone requires a dozen separate protein cofactors. In prokaryotes, for each of the three steps of translation, three specialized protein cofactors perform specific (and in several cases necessary) functions.

In 1958, the term "ribosomes" was proposed for the cellular ribonucleic particles with a sedimentation coefficient ranging from 20S to lOOS. In bacteria there are about 15,000 to 20,000 ribosomes per cell, which corresponds to one quarter of the total cellular mass. rRNA represents 85% of the total mass of the cellular RNA.

The ribosome is a crucial player in the context of basic cellular processes since it serves to interpret the genetic code brought by the messenger RNA and build up the chain of amino acids delivered by transfer RNA, which in turn folds into fully functional proteins, an extremely complex process called translation. 9

The Ribosome is one of the greatest wonders of molecular nanotechnology ever devised by our amazing unfathomable creator.

David S. Goodsell Atomic Evidence Seeing the Molecular Basis of Life page 7
Researchers have been working for decades on this elusive subject, assembling information from many sources to build the detailed understanding we have today.Crystallography has revealed the inner secrets of the ribosome in glorious detail. For many years, researchers studied the individual proteins by crystallography, slowly building up a picture of the whole molecule. Th en, in 2000, three labs presented atomic structures of the intact ribosomal subunits. One major insight from these structures was the discovery that the ribosome is a ribozyme, with one particular nucleotide in the RNA catalyzing the proteinbuilding reaction. The structures also revealed how the small subunit positions the messenger RNA, the details of the tunnel where the newly synthesized protein exits from the construction site, and a host of other interesting details.

Koonin, the logic of chance, page 376
Breaking the evolution of the translation system into incremental steps, each associated with a biologically plausible selective advantage is extremely difficult even within a speculative scheme let alone experimentally. Speaking of ribosomes, they are so well structured that when broken down into their component parts by chemical catalysts (into long molecular fragments and more than fifty different proteins) they reform into a functioning ribosome as soon as the divisive chemical forces have been removed, independent of any enzymes or assembly machinery – and carry on working.  Design some machinery which behaves like this and I personally will build a temple to your name!

My comment: Fortunately, people that recognize the magnificence of the creator of the Ribosome, build him churches and temples all over the globe, and give HIM glory.

The smallest known cytoplasmic ribosome is found in prokaryotic cells; these ribosomes are about 2.5 MDa and contain more than 4000 nucleotides of RNA and greater than 50 proteins. 10

Translation is one of the most complex biological processes, involving diverse protein factors and enzymes as well as messenger and transfer RNAs. The sequence of the PTC is possibly the most relevant stretch of nucleic acid to be studied if one aims to understand the origin of life. Nowadays, it is a consensus that the ribosome should be understood as a prebiotic machine that predated the origin of cells. The contingent appearance of this ribozyme capable of binding amino acids together was crucial to both the initial emergence and further development of the phenomenon of life7

The ribosome is a ‘‘living fossil‘‘, a particle so central to all cellular processes that it has essentially become frozen in time, preserving many ancestral features in its molecular structure. 8

The origin of the ribosomal protein synthesis network is considered to be the singular defining event in the origin of cells and the Tree of Life 4

The ribosome is a multi-part machine responsible for translating the genetic instructions during the assembly of proteins. According to Craig Venter, a widely respected biologist, the ribosome is “an incredibly beautiful complex entity” which requires a minimum of 53 proteins. Bacterial cells may contain up to 100,000 ribosomes, and human cells may contain millions. Biologist Ada Yonath, who won the Nobel Prize for her work on ribosomes, observes that they are “ingeniously designed for their functions.”

The translation of the nucleotide sequence of an mRNA molecule into protein takes place in the cytosol on a large ribonucleoprotein assembly called a ribosome. Each amino acid used for protein synthesis is first attached to a tRNA molecule that recognizes, by complementary base-pair interactions, a particular set of three nucleotides (codons) in the mRNA. As an mRNA is threaded through a ribosome, its sequence of nucleotides is then read from one end to the other in sets of three according to the genetic code. To initiate translation, a small ribosomal subunit binds to the mRNA molecule at a start codon (AUG) that is recognized by a unique initiator tRNA molecule. A
large ribosomal subunit then binds to complete the ribosome and begin protein synthesis. During this phase, aminoacyl-tRNAs—each bearing a specific amino acid—bind sequentially to the appropriate codons in mRNA through complementary base-pairing between tRNA anticodons and mRNA codons. Each amino acid is added to the C-terminal end of the growing polypeptide in four sequential steps: aminoacyl-tRNA binding, followed by peptide bond formation, followed by two ribosome translocation steps. Elongation factors use GTP hydrolysis both to drive these reactions forward and to improve the accuracy of amino acid selection. The mRNA molecule progresses codon by codon through the ribosome in the 5ʹ-to-3ʹ direction until it reaches one of three stop codons. A release factor then binds to the ribosome, terminating translation and releasing the completed polypeptide. Eukaryotic and bacterial ribosomes are closely related, despite differences in the number and size of their rRNA and protein components. The rRNA has the dominant role in translation, determining the overall structure of the ribosome, forming the binding sites for the tRNAs, matching the tRNAs to codons in the mRNA, and creating the active site of the peptidyl transferase enzyme that links amino acids together during translation.


* Each cell contains around 10 million ribosomes, i.e. 7000 ribosomes are produced in the nucleolus each minute.
* Each ribosome contains around 80 proteins, i.e. more than 0.5 million ribosomal proteins are synthesized in the cytoplasm per minute.
* The nuclear membrane contains approximately 5000 pores. Thus, more than 100 ribosomal proteins are imported from the cytoplasm to the nucleus per pore and minute. At the same time 3 ribosomal subunits are exported from the nucleus to the cytoplasm per pore and minute.

The evidence from the ribosome
a. “Spontaneous formation of the unlocked state of the ribosome is a multi-step process.”
b. The L1 stalks of the ribosome bend, rotate and uncouple – undergoing at least four distinct stalk positions while each tRNA ratchets through the assembly tunnel.  At one stage, for instance, “the L1 stalk domain closes and the 30S subunit undergoes a counterclockwise, ratchet-like rotation” with respect to another domain of the factory.  This is not simple.  “Subunit ratcheting is a complex set of motions that entails the remodeling of numerous bridging contacts found at the subunit interface that are involved in substrate positioning.”
c.The enzyme machine that translates a cell’s DNA code into the proteins of life is nothing if not an editorial perfectionist…the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products… To their further surprise, the ribosome lets go of error-laden proteins 10,000 times faster than it would normally release error-free proteins, a rate of destruction that Green says is “shocking” and reveals just how much of a stickler (insisting) the ribosome is about high-fidelity protein synthesis. (Rachel Green, a Howard Hughes Medical Institute investigator and professor of molecular biology and genetics: The Ribosome: Perfectionist Protein-maker Trashes Errors, 2009)
4. Interactions between molecules are not simply matters of matching electrons with protons.  Instead, large structural molecules form machines with moving parts.  These parts experience the same kinds of forces and motions that we experience at the macro level: stretching, bending, leverage, spring tension, ratcheting, rotation and translocation.  The same units of force and energy are appropriate for both – except at vastly different levels.
5. Every day,  Every day, essays about molecular machines are giving more and more biomolecular details, many without mentioning evolution and giving details about the process of how these machines evolved. Ribosomes, however, are life essential, and a prerequisite to make the proteins which replicate DNA, hence, it had to emerge prior evolution could start. So its emergence cannot be explained by evolution.  
6. These complexities are best explained by the work of an intelligent agency.
7. Hence, most probably, God exists.

Comparative genomic reconstructions of the gene repertoire of LUCA(S) point to a complex translation system that includes at least 18 of the 20 aminoacyl-tRNA synthetases (aaRS), several translation factors, at least 40 ribosomal proteins, and several enzymes involved in rRNA and tRNA modification. It appears that the core of the translation system was already fully shaped in LUCA(S) (Anantharaman, et al., 2002).

By the time of LUCA, the ribosome clearly exists in essentially its modern form.Thus, laboratory reconstructions will be needed. However, there would be limited value in resurrecting the complete ribosome of LUCA, because it was in effect a modern ribosome itself.
http://cshperspectives.net/content/2/9/a003483.full

The RNA Message Is Decoded in Ribosomes
The synthesis of proteins is guided by information carried by mRNA molecules. To maintain the correct reading frame and to ensure accuracy (about 1 mistake every 10,000 amino acids), protein synthesis is performed in the ribosome, a complex catalytic machine made from more than 50 different proteins (the ribosomal proteins) and several RNA molecules, the ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of ribosomes in its cytoplasm



The large and small ribosome subunits are assembled at the nucleolus, where newly transcribed and modified rRNAs associate with the ribosomal proteins that have been transported into the nucleus after their synthesis in the cytoplasm. These two ribosomal subunits are then exported to the cytoplasm, where they join together to synthesize proteins. Eukaryotic and bacterial ribosomes have similar structures and functions, being composed of one large and one small subunit that fit together to form a complete ribosome with a mass of several million daltons


The small subunit provides the framework on which the tRNAs are accurately matched to the codons of the mRNA, while the large subunit catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide chain (see Figure above). When not actively synthesizing proteins, the two subunits of the ribosome are separate. They join together on an mRNA molecule, usually near its 5ʹ end, to initiate the synthesis of a protein. The mRNA is then pulled through the ribosome, three nucleotides at a time. As its codons enter the core of the ribosome, the mRNA nucleotide sequence is translated into an amino acid sequence using the tRNAs as adaptors to add each amino acid in the correct sequence to the growing end of the polypeptide chain. When a stop codon is encountered, the ribosome releases the finished protein, and its two subunits separate again. These subunits can then be used to start the synthesis of another protein on another mRNA molecule. Ribosomes operate with remarkable efficiency: in one second, a eukaryotic ribosome adds 2 amino acids to a polypeptide chain; the ribosomes of bacterial cells operate even faster, at a rate of about 20 amino acids per second. To choreograph the many coordinated movements required for efficient translation, a ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A site, the P site, and the E site) are for tRNAs


The RNA-binding sites in the ribosome. 
Each ribosome has one binding site for mRNA and three binding sites for tRNA: the A, P, and E sites (short for aminoacyl-tRNA, peptidyl-tRNA, and exit, respectively). 
(A) A bacterial ribosome viewed with the small subunit in the front (dark green) and the large subunit in the back (light green). Both the rRNAs and the ribosomal proteins are illustrated. tRNAs are shown bound in the E site (red), the P site (orange), and the A site (yellow). Although all three tRNA sites are shown occupied here, during the process of protein synthesis not more than two of these sites are thought to contain tRNA molecules at any one time. 
(B) Large and small ribosomal subunits arranged as though the ribosome in (A) were opened like a book. 
(C) The ribosome in (A) rotated through 90° and viewed with the large subunit on top and small subunit on the bottom. (D) Schematic representation of a ribosome [in the same orientation as (C)], which will be used
in subsequent figures.

A tRNA molecule is held tightly at the A and P sites only if its anticodon forms base pairs with a complementary codon (allowing for wobble) on the mRNA molecule that is threaded through the ribosome. 


The path of mRNA (blue) through the small ribosomal subunit

The A andP sites are close enough together for their two tRNA molecules to be forced to form base pairs with adjacent codons on the mRNA molecule. This feature of the ribosome maintains the correct reading frame on the mRNA.


Comment: that means, in order to maintain the correct reading frame on the mRNA, the configuration of the A and P sites to be close enough together IS VITAL. How could this configuration have emerged randomly? Trial and error ? This tiny fact means, there is no tolerance here. It is an all or nothing business. The configuration HAS TO BE RIGHT just from the beginning. A down up development to get the right distance will be always non-functional. Another important evidence that demonstrates that evolutionary means are not adequate to explain the feat in question.   


Once protein synthesis has been initiated, each new amino acid is added to the elongating chain in a cycle of reactions containing four major steps: 

tRNA binding (step 1), 
peptide bond formation (step 2), 
large subunit translocation (step 3), 
and small subunit translocation (step 4). 

As a result of the two translocation steps, the entire ribosome moves three nucleotides along the mRNA and is positioned to start the next cycle.

Comment: this is a process that only works in a fully developed arrangement and sequence. If ANY of the four steps is missing, no deal. The machinelike  polymerization process will not work.  


Translating an mRNA molecule. 
Each amino acid added to the growing end of a polypeptide chain is selected by complementary basepairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain. Because only one of the many types of tRNA molecules in a cell can base-pair with each codon, the codon determines the specific amino acid to be added to the growing polypeptide chain. The four-step cycle shown is repeated over and over during the synthesis of a protein. In step 1, an aminoacyl-tRNA molecule binds to a vacant A site on the ribosome. In step 2, a new peptide bond is formed. In step 3, the large subunit translocates relative to the small subunit, leaving the two tRNAs in hybrid sites: P on the large subunit and A on the small, for one; E on the large subunit and P on the small, for the other. In step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome. This “resets” the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA molecule to bind. As indicated, the mRNA is translated in the 5ʹ-to-3ʹ direction, and the N-terminal end of a protein is made first, with each cycle adding one amino acid to the C-terminus of the polypeptide chain

Comment:  proof reading is required for getting an error rate which keeps the mutation rates sufficiently low.  Elongation factors EF-Tu and EF-G are part of the toolkit providing that error reduction. This is clear evidence of a designed process. Without the mechanisms to provide that error reduction, there would be no life. 

The image above illustrates this four-step process, beginning at a point at which three amino acids have already been linked together and there is a tRNA molecule in the P site on the ribosome, covalently joined to the C-terminal end of the short polypeptide. In step 1, a tRNA carrying the next amino acid in the chain binds to the ribosomal A site by forming base pairs with the mRNA codon positioned there, so that the P site and the A site contain adjacent bound tRNAs. In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by breakage of the high-energy bond between the tRNA and its amino acid) and joined to the free amino group of the amino acid linked to the tRNA at the A site, forming a new peptide bond. This central reaction of protein synthesis is catalyzed by a peptidyl transferase contained in the large ribosomal subunit. In step 3, the large subunit moves relative to the mRNA held by the small subunit, thereby shifting the acceptor stems of the two tRNAs to the E and P sites of the large subunit. In step 4, another series of conformational changes moves the small subunit and its bound mRNA exactly three nucleotides, ejecting the spent tRNA from the E site and resetting the ribosome so it is ready to receive the next aminoacyl-tRNA. Step 1 is then repeated with a new incoming aminoacyl-tRNA, and so on. This four-step cycle is repeated each time an amino acid is added to the polypeptide chain, as the chain grows from its amino to its carboxyl end.

Elongation Factors Drive Translation Forward and Improve Its Accuracy
The basic cycle of polypeptide elongation shown in outline in Figure above has an additional feature that makes translation especially efficient and accurate. Two elongation factors enter and leave the ribosome during each cycle, each hydrolyzing GTP to GDP and undergoing conformational changes in the process. These factors are called EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes. Under some conditions in vitro, ribosomes can be forced to synthesize proteins without the aid of these elongation factors and GTP hydrolysis, but this synthesis is very slow, inefficient, and inaccurate. Coupling the GTP hydrolysis-driven changes in the elongation factors to transitions between different states of the ribosome speeds up protein synthesis enormously. The cycles of elongation factor association, GTP hydrolysis, and dissociation also ensure that all such changes occur in the “forward” direction, helping translation to proceed efficiently


Detailed view of the translation cycle. 
Shown are the roles of the two elongation factors EF-Tu and EF-G, which drive translation in the forward direction. EF-Tu provides opportunities for proofreading of the codon– anticodon match. In this way, incorrectly paired tRNAs are selectively rejected, and the accuracy of translation is improved. The binding of a molecule of EF-G to the ribosome and the subsequent hydrolysis of GTP lead to a rearrangement of the ribosome structure, moving the mRNA being decoded exactly three nucleotides through it

In addition to moving translation forward, EF-Tu increases its accuracy.  EF-Tu can simultaneously bind GTP and aminoacyl-tRNAs, and it is in this form that the initial codon–anticodon interaction occurs in the A site of the ribosome. Because of the free-energy change associated with base-pair formation, a correct codon–anticodon match will bind more tightly than an incorrect interaction. However, this difference in affinity is relatively modest and cannot by itself account for the high accuracy of translation. To increase the accuracy of this binding reaction, the ribosome and EF-Tu work together in the following ways. First, the 16s rRNA in the small subunit of the ribosome assesses the “correctness” of the codon–anticodon match by folding around it and probing its molecular details


Recognition of correct codon–anticodon matches by the small-subunit rRNA of the ribosome. 
Shown here is the interaction between a nucleotide of the small-subunit rRNA and the first nucleotide pair of a correctly paired codon–anticodon. Similar interactions form between other nucleotides of the rRNA and the second and third positions of codon– anticodon pair. The small-subunit rRNA can form this network of hydrogen bonds only when an anticodon is correctly matched to a codon. As explained in the text, this codon–anticodon monitoring by the small-subunit rRNA increases the accuracy of protein synthesis.


Recognition of Cognate Transfer RNA by the 30S Ribosomal Subunit
https://sci-hub.st/https://science.sciencemag.org/content/292/5518/897

The ribosome recognizes the geometry of codonanticodon base pairing in a way that would discriminate against near-cognate tRNAs. The minor groove of the first and second base pairs between the codon and anticodon is closely monitored by a set of interactions that are induced by the binding of cognate tRNA. These interactions would be disrupted by mismatches, so that the induced structural changes would no longer be energetically favorable. The third or “wobble” position has less stringent constraints, and therefore can allow a broader range of base-pairing geometries, consistent with the requirements of the genetic code. The binding of paromomycin partially induces the changes that normally are induced by cognate tRNA. 

Selection of tRNA by the Ribosome Requires a Transition from an Open to a Closed Form NOVEMBER 27, 2002
https://www.cell.com/fulltext/S0092-8674(02)01086-3
The selection by the ribosome of substrate aminoacyl-transfer RNAs (aa-tRNAs) for incorporation of amino acids into a growing peptide chain depends on base complementarity between the codon on mRNA and the anticodon on tRNA. However, near-cognate tRNAs, which differ from the correct or cognate tRNAs by a single, subtle mismatch in codon-anticodon base-pairing, cannot be accurately discriminated against on the basis of differences in the free energy of base-pairing alone.  The ribosome recognizes base-pairing geometry during decoding, thus raising the intrinsic selectivity of each step to the levels required. The structure of the 30S subunit with A site codon and cognate tRNA anticodon stem loop (ASL) subsequently revealed conformational changes in the 30S, in which A1492, A1493, and G530 interact intimately with the minor groove of the first two codon-anticodon base pairs For any given codon in the A site, the ribosome must be able to distinguish cognate tRNA from all others, in a sequence-independent manner.

Both the 30S subunit and polymerases undergo a rearrangement from an open to a closed form, which interacts intimately with the minor groove of Watson-Crick substrate base pairs. Thus, a common mechanistic principle ensures accurate complementary base-pairing during replication, transcription, and translation of the genetic information.

Before an amino acid is added to a growing polypeptide chain, the ribosome folds around the codon–anticodon interaction, and only when the match is correct is this folding completed and the reaction allowed to proceed. Thus, the codon–anticodon interaction is thereby checked twice—once by the initial complementary base-pairing and a second time by the folding of the ribosome, which depends on the correctness of the match. This same principle of induced fit is seen in transcription by RNA polymerase; here, an incoming nucleoside triphosphate initially forms a base pair with the template; at this point the enzyme folds around the base pair (thereby assessing its correctness) and, in doing so, creates the active site of the enzyme. The enzyme then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being incorporated into the growing chain. A second principle used to increase the specificity of complementary base-pairing is called kinetic proofreading. We have seen that after the initial codon‒anticodon pairing and conformational change of the ribosome, GTP is hydrolyzed. This creates an irreversible step and starts the clock on a time delay during which the aminoacyl-tRNA moves into the proper position for catalysis. During this delay, those incorrect codon–anticodon pairs that have somehow slipped through the induced-fit scrutiny have a higher likelihood of dissociating than correct pairs. There are two reasons for this: (1) the interaction of the wrong tRNA with the codon is weaker, and (2) the delay is longer for incorrect than correct matches. kinetic proofreading thus increases the specificity of complementary base-pairing above what is possible from simple thermodynamic associations alone. The increase in specificity produced by kinetic proofreading comes at an energetic cost in the form of ATP or GTP hydrolysis. Kinetic proofreading is believed to operate in many biological processes, but its role is understood particularly well for translation.

The Ribosome Is a Ribozyme
The ribosome is a large complex composed of two-thirds RNA and one-third protein. The determination, in 2000, of the entire three-dimensional conformation of its large and small subunits is a major triumph of modern structural biology. The findings confirm earlier evidence that rRNAs—and not proteins—are responsible for the ribosome’s overall structure, its ability to position tRNAs on the mRNA, and its catalytic activity in forming covalent peptide bonds. The ribosomal RNAs are folded into highly compact, precise three-dimensional structures that form the compact core of the ribosome and determine its overall shape


Structure of the rRNAs in the large subunit of a bacterial ribosome, as determined by x-ray crystallography.
(A) Three-dimensional conformations of the large-subunit rRNAs (5S and 23S) as they appear in the ribosome. One of the protein subunits of the ribosome (L1) is also shown as a reference point, since it forms a characteristic protrusion on the ribosome.
(B) Schematic diagram of the secondary structure of the 23S rRNA, showing the extensive network of base-pairing. The structure has been divided into six “domains” whose colors correspond to those in (A). The secondarystructure diagram is highly schematized to represent as much of the structure as possible in two dimensions. To do this, several discontinuities in the RNA chain have been introduced, although in reality the 23S rRNA is a single RNA molecule. For example, the base of Domain III is continuous with the base of Domain IV even though a gap appears in the diagram.

In marked contrast to the central positions of the rRNAs, the ribosomal proteins are generally located on the surface and fill in the gaps and crevices of the folded RNA


Location of the protein components of the bacterial large ribosomal subunit.
The rRNAs (5S and 23S) are shown in blue and the proteins of the large subunit in green. This view is toward the outside of the ribosome; the interface with the small subunit is on the opposite face.

Some of these proteins send out extended regions of polypeptide chain that penetrate short distances into holes in the RNA core


L15 protein in the large subunit of the bacterial ribosome. 
The globular domain of the protein lies on the surface of the ribosome and an extended region penetrates deeply into the RNA core of the ribosome. The L15 protein is shown in green and a portion of the ribosomal RNA core is shown in blue.

The main role of the ribosomal proteins seems to be to stabilize the RNA core, while permitting the changes in rRNA conformation that are necessary for this RNA to catalyze efficient protein synthesis. 

My comment: There is clear teleological meaning here. Why would random events produce these proteins with a distant gole ( to stabilize the RNA core ) , if they are only becoming functional, once the ribosome multiprotein macromolecule holocomplex is fully assembled and working in an integrated fashion? 

The proteins also aid in the initial assembly of the rRNAs that make up the core of the ribosome.

My comment: Foreknowledge is required to know that tRNA's are required in the process of translation, and helping in the biosynthesis of those. From a naturalistic/evolutionary standpoint, it would make no sense whatsoever to predict that ribosome proteins would be helping in assembling other proteins. 

Not only are the A, P, and E binding sites for tRNAs formed primarily by ribosomal RNAs, but the catalytic site for peptide bond formation is also formed by RNA, as the nearest amino acid is located more than 1.8 nm away. This discovery came as a surprise to biologists because, unlike proteins, RNA does not contain easily ionizable functional groups that can be used to catalyze sophisticated reactions like peptide bond formation. Moreover, metal ions, which are often used by RNA molecules to catalyze chemical reactions, were not observed at the active site of the ribosome. Instead, it is believed that the 23S rRNA forms a highly structured pocket that, through a network of hydrogen bonds, precisely orients the two reactants (the growing peptide chain and an aminoacyl-tRNA) and thereby greatly accelerates their covalent joining. An additional surprise came from the discovery that the tRNA in the P site contributes an important OH group to the active site and participates directly in the catalysis. This mechanism may ensure that catalysis occurs only when the P site tRNA is properly positioned in the ribosome. RNA molecules that possess catalytic activity are known as ribozymes. We saw earlier in this chapter that some ribozymes function in self-splicing reactions.


 




Two massive polymolecular units that combine to make the ribosome are needed for translation. The small unit is on the left and the large unit is on the right. Combined they represent 4 large RNA molecules with 70 proteins attached to the frame. The unit is a masterpiece of precision engineering… not a random association of stuff. 6


Recognition of Cognate Transfer RNA by the 30S Ribosomal Subunit
https://sci-hub.st/https://science.sciencemag.org/content/292/5518/897


The ribosome recognizes the geometry of codon anticodon base pairing in a way that would discriminate against near-cognate tRNAs. The minor groove of the first and second base pairs between the codon and anticodon is closely monitored by a set of interactions that are induced by the binding of cognate tRNA. These interactions would be disrupted by mismatches, so that the induced structural changes would no longer be energetically favorable. The third or “wobble” position has less stringent constraints, and therefore can allow a broader range of base-pairing geometries, consistent with the requirements of the genetic code. 

My comment: Monitoring and taking action when something is wrong requires "knowledge" of the correct state, "knowledge" of the wrong state, and know how to correct the wrong state. Knowledge, monitoring, error recognition and repair are actions only known to be performed by intelligence. 






A GIF animation of how the ribosome works:
https://upload.wikimedia.org/wikipedia/commons/9/94/Protein_translation.gif

https://www.youtube.com/watch?v=Z2XOhgRJVb4&t=311s
https://www.youtube.com/watch?v=5_64XkJeSLU
https://www.youtube.com/watch?v=C4QiMqBSDe4

EF-Tu delivers aminoacyl-tRNA to the ribosome

A. E. Dahlberg - Ribosome structure and function
https://www.youtube.com/watch?v=2guXr5c4rHc

animation:
http://telstar.ote.cmu.edu/biology/animation/ProteinSynthesis/proteinsynthesis.html

The Crystal Structure Of The 50s Large Ribosome
https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html?&mmdbid=22801&bu=1&showanno=1&source=full-feature

1. from the book: The Logic of Chance: The Nature and Origin of Biological Evolution , page 228, By Eugene V. Koonin
2. http://www.sciencemag.org/news/2017/06/there-are-millions-protein-factories-every-cell-surprise-they-re-not-all-same
3. http://www.nobelprize.org/educational/medicine/dna/a/translation/ribosome_ass.html
4. https://www.sciencedirect.com/science/article/pii/S0040580918300789
5. https://sci-hub.tw/https://www.nature.com/articles/nrm2352
6. https://blueprintsforliving.com/cellular-ribosomes-the-origin-of-life/
7. https://jcs.biologists.org/content/117/17/3725
8. https://pubmed.ncbi.nlm.nih.gov/28454764/
9. https://sci-hub.st/https://www.sciencedirect.com/science/article/pii/B9780081022689000057
10. [url=https://onlinelibrary.wiley.com/doi/abs/10.1002/bip.10221]https://onlinelib

Accuracy in Translation Requires an Expenditure of Free Energy
Translation by the ribosome is a compromise between the opposing constraints of accuracy and speed. We have seen, for example, that the accuracy of translation (1 mistake per 104 amino acids joined) requires time delays each time a new amino acid is added to a growing polypeptide chain, producing an overall speed of translation of 20 amino acids incorporated per second in bacteria. Mutant bacteria with a specific alteration in the small ribosomal subunit have longer delays and translate mRNA into protein with an accuracy considerably higher than this; however, protein synthesis is so slow in these mutants that the bacteria are barely able to survive.

Question: How could the right speed have been obtained with trial and error, if slow mutants do not survive? Had the speed not to be right right from the beginning?

We have also seen that attaining the observed accuracy of protein synthesis requires the expenditure of a great deal of free energy; this is expected, since, there is a price to be paid for any increase in order in the cell. In most cells, protein synthesis consumes more energy than any other biosynthetic process. At least four high-energy phosphate bonds are split to make each new peptide bond: two are consumed in charging a tRNA molecule with an amino acid, and two more drive steps in the cycle of reactions occurring on the ribosome during protein synthesis itself. In addition, extra energy is consumed each time that an incorrect amino acid linkage is hydrolyzed by a tRNA synthetase  and each time that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected. To be effective, any proofreading mechanism must also allow an appreciable fraction of correct interactions to be removed; for this reason, proofreading is even more costly in energy than it might at first seem.

My comment: So we see the same proofreading principles applied in totally distinct protein complexes. Furthermore, proof reading requires energy.  This is, therefore, clear evidence of design. 


Translation
http://europepmc.org/article/med/19595805#R18
The ribosome “reads” mRNA in a 5’-3’ direction and synthesizes the corresponding protein from its N-terminus. Both ribosomal subunits contain three binding sites for tRNA molecules that are in distinct functional states:

(i) the A site binds the aminoacyl-tRNA, which brings a new amino acid that is to be incorporated into the growing polypeptide,
(ii) the P site binds the peptidyl-tRNA (as well as the initiator fMet-tRNAfmet), and
(iii) the E site binds the deacylated tRNA that is to be soon dissociated from the ribosome.

The 30S subunit binds mRNA and it ensures the fidelity of translation through close monitoring of the anticodon-codon interactions. The 50S subunit, on the other hand, binds the acceptor ends of substrate tRNAs and it catalyzes the peptide bond formation in which an α-amino group of the aminoacyl-tRNA attacks the carbonyl carbon of the peptidyl-tRNA. From a chemical standpoint the reaction is an aminolysis of an acyl-ester link formed between the carbonyl carbon of the peptidyl moiety and the O3’ atom of the P-site A76. Upon peptide bond synthesis, the lengthened peptidyl-tRNA is bound to the A site, whereas the deacylated tRNA is in the P site. Peptide elongation is further promoted by the GTP-dependent protein elongation factors EF-G and EF-Tu. EF-G promotes translocation of the A-site bound peptidyl-tRNA into the P site and of the P-site bound deacylated tRNA into the E site. Consequently, the ribosome moves down the mRNA filament with an active site that is ready for a new reaction cycle. Then, the elongation factor EF-Tu delivers the aminoacyl-tRNA to the A site. The proper codon-anticodon interactions stimulate the GTP-ase activity of EF-Tu, leading to its dissociation from the complex. The acceptor end of the A-site aminoacyl-tRNA reorients and positions an incoming amino acid for the reaction with the peptidyl moiety attached to the P-site peptidyl-tRNA in a process known as accommodation. The rate of accommodation is significantly slower than the rate of peptide bond formation and is measured to be ~10s−1. This rate is, however, much higher when small analogs are used and is estimated to be ~50s−1 allowing detailed kinetic studies using this system.

Question: How could this finely tuned and precise orchestration have emerged throug a stepwise evolutionary process, in face of the fact that ALL players have an essential role in the peptyde bond formation and polymer elongation process? 

The Ribosomal Peptidyl Transferase Center: Structure, Function, Evolution, Inhibition
https://geneticacomportamento.ufsc.br/files/2013/08/Polacek05-Ribozima-peptidil-transferase.pdf
The distinctive features of the modern ribosome are its mammoth size and enormous structural complexity. The molecular weight of the ribosome exceeds 2.5 million daltons and the particle comprises at least three large rRNA molecules and more than 50 different ribosomal proteins. It is unimaginable that the ribosome, which contains rRNAs of thousands of nucleotides in length, evolved in a single evolutionary step.

Crystal Structure Of The 30s Ribosomal Subunit From Thermus Thermophilus In Complex With The Translation Initiation Factor If3 (C-Terminal Domain)
https://www.ncbi.nlm.nih.gov/Structure/pdb/1I96



There are millions of protein factories in every cell. Surprise, they’re not all the same 2

The plant that built your computer isn't churning out cars and toys as well. But many researchers think cells' crucial protein factories, organelles known as ribosomes, are interchangeable, each one able to make any of the body's proteins. Now, a provocative study suggests that some ribosomes, like modern factories, specialize in manufacturing only certain products. Such tailored ribosomes could provide a cell with another way to control which proteins it generates. They could also help explain the puzzling symptoms of certain diseases, which might arise when particular ribosomes are defective.

Biologists have long debated whether ribosomes specialize, and some remain unconvinced by the new work. But other researchers say they are sold on the finding, which relied on sophisticated analytical techniques. "This is really an important step in redefining how we think about this central player in molecular biology," says Jonathan Dinman, a molecular biologist at the University of Maryland in College Park.

A mammalian cell may harbor as many as 10 million ribosomes, and it can devote up to 60% of its energy to constructing them from RNA and 80 different types of proteins. Although ribosomes are costly, they are essential for translating the genetic code, carried in messenger RNA (mRNA) molecules, into all the proteins the cell needs. "Life evolved around the ribosome," Dinman says.

The standard view has been that a ribosome doesn't play favorites with mRNAs—and therefore can synthesize every protein variety. But for decades, some researchers have reported hints of customized ribosomes. For example, molecular and developmental biologist Maria Barna of Stanford University in Palo Alto, California, and colleagues reported in 2011 that mice with too little of one ribosome protein have short tails, sprout extra ribs, and display other anatomical defects. That pattern of abnormalities suggested that the protein shortage had crippled ribosomes specialized for manufacturing proteins key to embryonic development.

Definitive evidence for such differences has been elusive, however. "It's been a really hard field to make progress in," says structural and systems biologist Jamie Cate of the University of California (UC), Berkeley. For one thing, he says, measuring the concentrations of proteins in naturally occurring ribosomes has been difficult.

In their latest study, published online last week in Molecular Cell, Barna and her team determined the abundances of various ribosome proteins with a method known as selected reaction monitoring, which depends on a type of mass spectrometry, a technique for sorting molecules by their weight. When the researchers analyzed 15 ribosomal proteins in mouse embryonic stem cells, they found that nine of the proteins were equally common in all ribosomes. However, four were absent from 30% to 40% of the organelles, suggesting that those ribosomes were distinctive. Among 76 ribosome proteins the scientists measured with another mass spectrometry-based method, seven varied enough to indicate ribosome specialization.

Barna and colleagues then asked whether they could identify the proteins that the seemingly distinctive ribosomes made. A technique called ribosome profiling enabled them to pinpoint which mRNAs the organelles were reading—and thus determine their end products. The specialized ribosomes often concentrated on proteins that worked together to perform particular tasks. One type of ribosome built several proteins that control growth, for example. A second type churned out all the proteins that allow cells to use vitamin B12, an essential molecule for metabolism. That each ribosome focused on proteins crucial for a certain function took the team by surprise, Barna says. "I don't think any of us would have expected this."

Ribosome specialization could explain the symptoms of several rare diseases, known as ribosomopathies, in which the organelles are defective. In Diamond-Blackfan anemia, for instance, the bone marrow that generates new blood cells is faulty, but patients also often have birth defects such as a small head and misshapen or missing thumbs. These seemingly unconnected abnormalities might have a single cause, the researchers suggest, if the cells that spawn these different parts of the body during embryonic development carry the same specialized ribosomes.

Normal cells might be able to dial protein production up or down by adjusting the numbers of these specialized factories, providing "a new layer of control of gene expression," Barna says. Why cells need another mechanism for controlling gene activity isn't clear, says Cate, but it could help keep cells stable if their environment changes.


The ribosome in a ‘nutshell’ 
The human 80S ribosome is composed of a 40S and a 60S subunit. The chief function of the 40S subunit, which contains one strand of 18S ribosomal RNA (rRNA) and 33 distinct RPs, is to bind, unwind and scan mRNAs, whereas the 60S subunit is responsible for peptide bond formation and quality control of nascent peptides14 and is composed of three strands of rRNA (5S, 5.8S and 28S rRNA) and 47 distinct RPs15. The nucleolus is the main site of ribosome biogenesis and forms around nucleolar organizer regions (NORs), which contain several hundred ribosomal DNA (rDNA) gene repeats in human diploid cells16–18. These repeats are generally arranged head-to-tail in tandem with a number of palindromic units19 and are all located in five clusters on the short arms of acrocentric chromosomes 13, 14, 15, 21 and 22
https://sci-hub.ren/10.1038/nrc.2017.104

In eukaryotes, translation initiation results in the recruitment of the 40S ribosomal subunit (SSU), initiation factors and the initiator Met-tRNAi Met to the start codon, followed by joining of the 60S ribosomal subunit to form the 80S ribosome.


Ribosomes are large molecular machines the catalyze the translation of genetic information into proteins, one of the most fundamental processes in every organism. They consist of a small and a large subunit, both of which are composed of ribosomal RNA (rRNA) and ribosomal proteins. The atomic X-ray structures of bacterial ribosomes that emerged in the early 2000s helped understanding many fundamental aspects of the translation process and provided fascinating new functional insights into bacterial translation in particular. In 2009, this pioneering work was awarded with the Nobel Prize in Chemistry 10


Experimental views of a bacterial ribosome.
The upper image shows a 3D reconstruction from electron microscopy, with the small subunit in green and the large subunit in blue . The lower image is an atomic structure from x-ray crystallography and an NMR structure of a fl exible protein stalk that is not observed in the crystal structure

More Non-Random DNA Wonders
https://web.archive.org/web/20130406102730/http://iaincarstairs.wordpress.com/2011/12/26/more-non-random-dna-wonders/

keggs pathway





An overview of ribosomal structure and mRNA translation.
mRNA translation is initiated with the binding of tRNA to the P site . An incoming tRNA is delivered to the A site in complex with elongation factor (EF)-Tu–GTP. Correct codon–anticodon pairing activates the GTPase centre of the ribosome, which causes hydrolysis of GTP and release of the amino acyl end of the tRNA from EF-Tu. Binding of tRNA also induces conformational changes in ribosomal (r)RNA that optimally orientates the peptidyl-tRNA and aminoacyl-tRNA for the peptidyl-transferase reaction to occur, which involves the transfer of the peptide chain onto the A-site tRNA. The ribosome must then shift in the 3′ mRNA direction so that it can decode the next mRNA codon. Translocation of the tRNAs and mRNA is facilitated by binding of the GTPase EF-G, which causes the deacylated tRNA at the P site to move to the E site and the peptidyl-tRNA at the A site to move to the P site upon GTP hydrolysis. The ribosome is then ready for the next round of elongation. The deacylated tRNA in the E site is released on binding of the next aminoacyl-tRNA to the A site. Elongation ends when a stop codon is reached, which initiates the termination reaction that releases the polypeptide 5


A schematic diagram of the elongation phase of the ribosome-catalyzed translation.
A peptidyl-tRNA is bound to the P site and the deacylated tRNA is in the ribosomal E site. The elongation factor EF-Tu complexed with GTP (orange) delivers an aminoacyl-tRNA to the A site. The deacylated tRNA dissociates from the E site on binding of the aminoacyl-tRNA to the A site. Upon codon recognition the GTP-ase activity of EF-Tu is stimulated and this causes a conformational change in EF-Tu upon which the factor dissociates from the ribosome. If the appropriate codon-anticodon interaction is established the CCA-end of the A-site aminoacyl-tRNA undergoes conformational change in a process known as accommodation, whereas the non-cognate tRNA is rejected at this point. After accommodation a free α-amino group of the aminoacyl-tRNA is oriented properly for the nucleophilic attack onto the acyl-ester link of the peptidyl-tRNA in the P site. The peptidyl transfer reaction occurs rapidly yielding a lengthened peptidyl-tRNA bound to the A site and the deacylated tRNA in the P site. The translocation of the reaction products and mRNA is promoted by the elongation factor EF-G in a GTP-dependent manner. The peptidyl-tRNA moves from the A site into the P site, whereas the deacylated tRNA moves from the P site into the E site. Also, the ribosome has now shifted in the 3’ direction of the mRNA and a new codon occupies the A site on the 30S subunit. After dissociation of the EF-G:GDP complex from the ribosome a new round of peptide synthesis ensues. Once the ribosome encounters the translational stop codon the termination phase of protein synthesis is initiated (not shown).


An overview of termination of translation. 
A stop codon in the mRNA A site (red hexagon) recruits either release factor-1 (RF1) or RF2 to mediate the hydrolysis and release of the peptide from the tRNA in the P site. This functions as a signal to recruit RF3–GDP, which induces the release of RF1/2. Exchange of GDP for GTP on RF3 and subsequent hydrolysis is thought to release RF3. The ribosome is left with mRNA and a deacylated tRNA in the P site. This complex is disassembled by the binding of ribosomal release factor (RRF) and the EF-G elongation factor62. GTP hydrolysis causes the dissociation of the 50S ribosomal subunit, and initiation factor-3 (IF3) is required to dissociate the deacylated tRNA from the P site.


Ribosomes in action.
Three atomic structures capture ribosomes ( blue and green ) in the process of building a protein chain. Elongation factor Tu ( magenta ) delivers a new transfer RNA ( yellow ), pairing its anticodon with the messenger RNA ( red ) codon. The ribosome then catalyzes the formation of the peptide bond; the structure at the center includes two transfer RNA molecules with amino acids attached ( bright green ) and positioned in the catalytic
site. Finally, elongation factor G ( magenta ) shifts everything by one codon (toward the right in this illustration), opening a space for the next transfer RNA

Trans-Translation

The final step of the central dogma is the most complex, where the information in RNA is translated to build proteins. A constellation of proteins and specialized RNA molecules are needed to prepare and deliver the amino acids needed for each step, and researchers have studied them one by one, filling out all the pieces to this biomolecular puzzle.  Ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for
instance, for a messenger RNA that has broken and is thus is missing its stop codon. A special mechanism is used to rescue these stalled ribosomes when they get stuck at the end of the truncated chain. A strangely shaped RNA molecule mimics both a transfer RNA and a messenger RNA, restarting the process and cleaning up the mess.


Transfer-messenger RNA.
Transfer-messenger RNA (top ) includes a portion that mimics a transfer RNA ( red ) and a portion that mimics a messenger RNA ( magenta ), complete with a stop codon. It binds to stalled ribosomes ( bottom ), resuming synthesis using its own short message. Amazingly, this message encodes a small tag that is added to the end of the truncated protein, signaling to the cell that the protein is faulty and needs to be destroyed

My comment:  This is awesome. Wow!! How could random non-guided, non-intelligent mechanisms have foresight, and the knowledge of this problem of truncated messenger RNA's that do not have a stop codon, and a sophisticated mechanism to deal with it? This is a masterfully crafted salvage mechanism in order to prevent the cell to produce toxic polymer strands that would accumulate, and in the end, destroy the cell. This is an engineering marvel of extraordinary sophistication.

Transfer-messenger RNA (abbreviated tmRNA ) is a bacterial RNA molecule with dual tRNA-like and messenger RNA-like properties. The tmRNA forms a ribonucleoprotein complex (tmRNP) together with 

Small Protein B (SmpB), 
Elongation Factor Tu (EF-Tu), and 
ribosomal protein S1. 

In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein biosynthesis, for example when reaching the end of a messenger RNA which has lost its stop codon. The tmRNA is remarkably versatile: it recycles the stalled ribosome, adds a proteolysis-inducing tag to the unfinished polypeptide, and facilitates the degradation of the aberrant messenger RNA

Mispairing of bases also plays an essential role during the synthesis of proteins. The 20 amino acids in proteins are encoded by triplet codons in DNA, along with a few codons used to specify the end of a protein. Doing the math, we see that there are 64 possible codons, so there is some degeneracy to the code, and several different codons are used to specify the same amino acid. However, if we look inside the nucleus, we find that there are only 20 or so types of transfer RNA that match up the appropriate amino acids with its codon. This requires some mismatching of the transfer RNA anticodon with all of these different codons. This is accomplished
by allowing some “wobble” in the third position of the codon, so that different pairings are allowed. When the structures of ribosomes were solved, it was found that the first two bases in the codon are tightly controlled by the ribosome, ensuring only the proper pairing, but the third position is looser, allowing some wobble

Catalytic mechanism
The ribosome catalyzes an aminolysis of an acyl-ester bond in the P site. The reaction begins with a nucleophilic attack of the α-amino group of the aminoacyl-tRNA bound in the A site onto the carbonyl carbon of the peptidyl-tRNA positioned in the P site and it proceeds through a tetrahedral oxyanion intermediate. The intermediate collapses into the lengthened peptidyl-tRNA in the A site and the deacylated tRNA in the P site. The ribosome brings ~10^7, or 10,000,000 ( 10 million )-fold increase in the reaction rate compared to an uncatalyzed reaction in solution

the ribosome utilizes a combination of the entropic, general acidbase and electrostatic shielding mechanisms to promote peptide bond formation. The two binding interfaces in the PTC, the A loop and the P loop, specifically recognize the acceptor ends of the aminoacyl- and the peptidyl-tRNA, respectively. The binding of the A-site substrate induces conformational change in the PTC, which allows reactive groups to be oriented properly for the peptidyl-transfer reaction (entropic component). Peptide bond formation is further promoted by a substrate-assisted mechanism in which the O2’ hydroxyl group of the P-site A76 plays a crucial role in the proton shuttling (general acid-base component), whereas a water molecule coordinated by the ribosomal bases stabilizes the oxyanion of the tetrahedral intermediate (electrostatic shielding component).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783306/

Peptide Bond Formation Mechanism Catalyzed by Ribosome
Peptide bond formation during protein polypeptide synthesis in the ribosome is one of the most important chemical reactions in life.  Peptide bond formation is of particular importance, being the heart of protein synthesis. 1 The process is so intriguingly complex, that a science paper in 2015 had still to admit that: The detailed mechanism of peptidyl transfer, as well as the atoms and functional groups involved in this process are still in limbo.
Another science paper confessed: The mechanism of this process and the origin of the catalytic power of this ancient enzyme are still an unsolved puzzle. As we know now, the process requires a precise and exquisitely engineered molecular arrangement, where one ribose molecule has the pivotal role as proton shuttle. 

Interactions between active site residues and the 2′-OH are pivotal in orienting substrates in the active site for optimal catalysis. A second 2′-OH group  was identified to be crucial for peptide bond formation, namely that of A2451. The 2′- OH of A2451 was shown to be of potential functional importance. 9

The ribosome promotes the reaction of the amino acid condensation by properly orienting the reaction substrates.

A2451, referring to adenine at position 2451 in the 23S rRNA of the large ribosomal subunit in bacteria (equivalent positions exist in eukaryotes within the 28S rRNA), plays a critical role in the function of the peptidyl transferase center (PTC) of the ribosome. This nucleotide is highly conserved across different species, underscoring its importance in the ribosome's catalytic activity. Its significance in the PTC can be understood in terms of its structural and functional contributions:

A2451 is strategically positioned within the PTC to contribute to the correct alignment and positioning of the aminoacyl-tRNA and peptidyl-tRNA within the ribosomal active site. This precise alignment is crucial for efficient and accurate peptide bond formation. A2451 helps in shaping the three-dimensional structure of the PTC, maintaining a catalytically active conformation that is essential for the ribosome's peptidyl transferase activity. While the PTC's catalytic mechanism is primarily ribozyme-based (RNA acting as an enzyme), the presence of A2451 is essential for facilitating the correct orientation of reactants and possibly stabilizing transition states during the peptide bond formation process. It's believed that A2451 and surrounding nucleotides contribute to the proper chemical environment necessary for catalysis, including the positioning of water molecules that may be involved in the reaction.
Interaction with Antibiotics: A2451 is also a binding site for several antibiotics that inhibit the PTC's function. For example, macrolide antibiotics bind near A2451, and their interaction can inhibit the translocation of the peptidyl-tRNA from the A-site to the P-site, effectively stopping protein synthesis. The sensitivity of A2451 to antibiotic binding highlights its functional significance within the PTC. Mutations at or near A2451 can lead to significant reductions in the peptidyl transferase activity of the ribosome, demonstrating the essential nature of this nucleotide for ribosomal function. Such studies provide direct evidence of A2451's critical role in the PTC. High-resolution crystal structures of the ribosomal large subunit have revealed the spatial arrangement of A2451 within the PTC, offering insights into its interactions with substrates and antibiotics, further confirming its essential role in the ribosome's function.
In summary, A2451 is vital for the PTC of the ribosome due to its contributions to the structural integrity and catalytic activity of the site. Its importance is highlighted by its evolutionary conservation, sensitivity to antibiotics, and the impact of mutations on ribosomal function, making it a key focus of research in understanding ribosome activity and the mechanism of protein synthesis.


Representation of 4-, 6- and 8-membered ring TS structures (A), ribosome structure (B) and schematic representation of the A and P site of Ribosome (C). Atoms included in the QM region are in shaded region. 2

A key role is given to the 2’ hydroxyl of the P site substrate, A76, by serving as a proton shuttle. It would act as a general base that would abstract the proton from the α-amino group and would donate a proton to the deacylated 3’ hydroxyl.

The full mechanism can be decomposed in three stages. These three stages take place in a single concerted step and they slightly overlap during the evolution of the system along the reaction path. The ribosome promotes an entropy trap, in the sense that it enhances the rate of the peptide bond formation positioning the substrates and/or excluding waters from the active site.

Key in the reaction is the presence of a proton shuttling group The observed 100-fold reduction in the reaction rate by mutation of P-site A76 20-OH group, however small, can be the indication of this group's activity during peptidyl transfer reaction.

Atomic mutagenesis suggests that nucleobases do not carry functional groups directly involved in peptide bond formation. Instead,A single ribose 20-hydroxyl group at A2451 was identified tobe of pivotal importance. 3


Ribosomal Protein Synthesis
Amide bond formation is the fundamental chemical reaction catalyzed by the PTC of the ribosome. The PTC possesses ribozyme activity: Attack of the a-amino group of the amino acid-charged A-site tRNA (orange) at the ester carbonyl carbon of the methionyl- or peptidyl-tRNA in the P site (blue) is supported to yield the amide linkage. The present study reveals details of this mechanism.

By using an atomic mutagenesis approach to investigate all the 23S rRNA residues that compose the inner core of the peptidyl transferase center, we identified a single functional group with crucial importance for peptide bond catalysis— namely, the ribose 20'-OH at A2451. This ribose 20 group needs to maintain hydrogen donor characteristics in order to promote effective amide bond formation. By combining these novel findings with biochemical and structural data that have accumulated over the last 2 decades highlighting the importance of the P-tRNA ribose 20'-OH at A76. The A2451 20 -hydroxyl directly assists in positioning the P-site tRNA-A76 ribose via hydrogen bond formation. This can promote an effective A76 ribose C20 -endo conformation to support amide synthesis via a proton shuttle mechanism. This synergistic approach appreciates the concept of ‘‘substrate-assisted catalysis’’ , and combines with it the strict functional requirement of the ribose 20 group at A2451 of 23S rRNA to possess hydrogen donor capability. 3

The A2451 2′-OH is central for establishing a proton wire 5.  In the proposed model the A2451 2′-OH is part of an array of H-donors/acceptors that subtracts a proton from the nucleophilic α-amine, and thereby facilitating the nucleophilic attack of the α-amine of the A-site aminoacyl-tRNA onto the ester carbonyl carbon of the P-site peptidyl-tRNA. As the intermediate breaks down to form a new peptide bond, the proton wire then relays a proton back to saturate the 3′-O− of the P-site tRNA leaving group. Evidently, the positioning of all substrates, transition states, and ribosomal residues contributing to the concerted redistribution of charges must be tightly controlled to achieve efficient transpeptidation compatible with the observed in vivo rates of amino acid polymerization of about 20 s–1.

This 2'-OH renders almost full catalytic power. These data highlight the unique functional role of the A2451 2'-OH for peptide bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site.

My comment: A precise, minutely orchestrated arrangement of just two main players,  the interaction of ribose 2'-OH at position A2451 , and the 2’ hydroxyl of the P site substrate A76  are pivotal in orienting substrates in the active site for optimal catalysis, and play a key role in polypeptide bond formation. The ribosome promotes the reaction of the amino acid condensation by properly orienting the reaction substrates. Key in the reaction is the presence of a proton shuttling group.  The observed 100-fold reduction in the reaction rate by mutation of P-site A76 20-OH group  is indication of this group's activity during the peptidyl transfer reaction.

The positioning of all substrates, transition states, and ribosomal residues contributing to the concerted redistribution of charges must be tightly controlled to achieve efficient transpeptidation. This 2'-OH renders almost full catalytic power. These data highlight the unique functional role of the A2451 2'-OH for peptide bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site.

Interactions between active site residues and the 2′-OH of A76 of the peptidyl-tRNA are pivotal in orienting substrates in this active site for optimal catalysis.

The 23S ribosomal RNA is 2904 nucleotides long (in E. coli) component of the large subunit (50S) of the bacterial/archean ribosome. In general, rRNA has an essential function of peptidyl transferase, the stimulating core of the ribosome plays role in the peptide bond configuration. Both peptidyl-tRNA and aminoacyl-tRNA are important for synthesizing protein and transpeptidation response. However, 23S rRNA positions which are G2252, A2451, U2506, and U2585 have a significant function for tRNA binding in P site of the large ribosomal subunit.

In the upper part of the tunnel, results suggest that A2062 and A2451 can communicate in both directions for translation stalling, mostly through dynamically coupled C2063, C2064, and A2450.

My comment:  This is truly awe-inspiring. The functional group A2451, which is not only of crucial importance as described above for peptyde bond catalysis, but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here", 

The large subunit 50S catalyzes peptide bond synthesis at the highly conserved catalytic cavity peptidyl transferase center (PTC), where nucleotides G2251, G2252, A2451, C2452, U2506, U2585, A2602 play critical roles in the translation process. 


Large subunit 50S, including P-tRNA (gray), polyAla chain (turquoise) and ribosomal proteins protruding to the ribosomal tunnel, namely uL4 (pink), uL22 (brown), uL23 (red) are shown. Nucleotides A2451 (PTC), A2062, U2585–U2586 (ribosomal tunnel), and residues Gly91 (tip of uL22 loop), Glu18 (trigger factor binding site on uL23), Gln72 (tip of uL23 loop), which are investigated in this study are also indicated. In all figures, PyMol (DeLano Scientific LLC., 2002) is used for the molecular visualization.

The ribosomal tunnel is not a passive passageway but is actively taking a role in translation regulation. The dynamics of the large subunit, the ribosomal tunnel geometry together with its electrostatic potential seems to play an important role on complexity and production rate of small folded proteins .  During its passage through the ribosomal tunnel, compacted chain interacts with the ribosomal tunnel elements and affects the recruitment of chaperones to the exit of the tunnel in bacteria. This suggests conformational crosstalk not only within the tunnel but also outside the tunnel at the solvent side.  This is literally a network of nucleotides and residues on the ribosomal tunnel taking a role in constant communication of the distant functional regions.

My comment:  What we see here, is a vivid and active masterfully performing information exchange taking place. The entire translation process is a carefully monitored, controlled, and regulated process where where information and signals are constantly being exchanged in order to guarantee a faithful and error minimized process of protein production. The key components that play in regulating the translation process are dispersed along the exit pathway.
 
1. https://sci-hub.st/https://pubs.rsc.org/en/content/articlelanding/2015/ra/c5ra02767e#!divAbstract
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582011/
3. https://core.ac.uk/download/pdf/81150349.pdf
4. https://en.wikipedia.org/wiki/23S_ribosomal_RNA
5. https://academic.oup.com/nar/article/46/4/1945/4788351
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116277/
7. https://www.frontiersin.org/articles/10.3389/fmolb.2020.586075/full
8. https://www.x-mol.com/paper/3338695?adv
9. https://sci-hub.st/https://pubs.acs.org/doi/full/10.1021/ja209558d
10. https://bangroup.ethz.ch/research/eukaryotic_ribosome.html

Ribosomes have several active centers
https://flylib.com/books/en/2.643.1.75/1/
The basic message to remember about the ribosome is that it is a cooperative structure that depends on changes in the relationships among its active sites during protein synthesis. The active sites are not small, discrete regions like the active centers of enzymes. They are large regions whose construction and activities may depend just as much on the rRNA as on the ribosomal proteins. The crystal structures of the individual subunits of bacterial ribosomes, and (at lesser resolution) of the intact ribosome, give us a good impression of the overall organization and emphasize the role of the rRNA.


Some sites in 16S rRNA are protected from chemical probes when 50S subunits join 30S subunits or when aminoacyl-tRNA binds to the A site. Others are the sites of mutations that affect protein synthesis. TERM suppression sites may affect termination at some or several termination codons. The large colored blocks indicate the four domains of the rRNA.

16S rRNA forms four general domains, in which just under half of the sequence is base-paired (see Figure above). The individual double-helical regions tend to be short (<8 bp). Often the duplex regions are not perfect, but contain bulges of unpaired bases. Comparable models have been drawn for mitochondrial rRNAs (which are shorter and have fewer domains) and for eukaryotic cytosolic rRNAs (which are longer and have more domains). The increase in length in eukaryotic rRNAs is due largely to the acquisition of sequences representing additional domains. Each domain of 16S rRNA folds independently and has a discrete location in the 30S subunit.


The ribosome has several active centers.
mRNA takes a turn as it passes through the A and P sites, which are angled with regard to each other. The E site lies beyond the P site. The peptidyl transferase site (not shown) stretches across the tops of the A and P sites. Part of the site bound by EF-Tu/G lies at the base of the A and P sites.

Much of the structure of the ribosome is occupied by its active centers. The expanded view of the ribosomal sites drawn in the Figure above shows they comprise about two-thirds of the ribosomal structure. A tRNA enters the A site, is transferred by translocation into the P site, and then leaves the (bacterial) ribosome by the E site. The A and P sites must extend across both ribosome subunits, since tRNA is paired with mRNA in the 30S subunit, but peptide transfer takes place in the 50S subunit. The A and P sites are adjacent, enabling translocation to move the tRNA from one site into the other. The problem of how two bulky tRNAs fit into the ribosome is solved by a turn in the path for mRNA. The E site is located near the P site (representing a position en route to the surface of the 50S subunit). The peptidyl transferase center is located on the 50S subunit, close to the aminoacyl ends of the tRNAs in the A and P sites.

All of the G-proteins that function in protein synthesis (EF-Tu, EF-G, IF-2, RF1,2,3) bind to a factor-binding site (sometimes called the GTPase center), which probably triggers their hydrolysis of GTP. It is located at the base of the stalk of the large subunit, which consists of the proteins L7/L12. (L7 is a modification of L12, and has an acetyl group on the N-terminus.) In addition to this region, the complex of protein L11 with a 58 base stretch of 23S rRNA provides the binding site for some antibiotics that affect GTPase activity. Neither of these ribosomal structures actually possesses GTPase activity, but they are both necessary for it. The role of the ribosome is to trigger GTP hydrolysis by factors bound in the factor-binding site.


Electron microscopic images of bacterial ribosomes and subunits reveal their shapes.

Initial binding of 30S subunits to mRNA requires protein S1, which has a strong affinity for single-stranded nucleic acid. It is responsible for maintaining the single-stranded state in mRNA that is bound to the 30S subunit. This action is necessary to prevent the mRNA from taking up a base-paired conformation that would be unsuitable for translation. S1 has an extremely elongated structure and associates with S18 and S21. The three proteins constitute a domain that is involved in the initial binding of mRNA and in binding initiator tRNA. This locates the mRNA-binding site in the vicinity of the cleft of the small subunit (see Figure above). The 3′ end of rRNA, which pairs with the mRNA initiation site, is located in this region.

The initiation factors bind in the same region of the ribosome. IF-3 can be crosslinked to the 3′ end of the rRNA, as well as to several ribosomal proteins, including those probably involved in binding mRNA. The role of IF-3 could be to stabilize mRNA P30S subunit binding; then it would be displaced when the 50S subunit joins.

The incorporation of 5S RNA into 50S subunits that are assembled in vitro depends on the ability of three proteins, L5, L8, and L25, to form a stoichiometric complex with it. The complex can bind to 23S rRNA, although none of the isolated components can do so. It lies in the vicinity of the P and A sites.

The important functional sites of the ribosome consists of both RNA and protein. At both the A site and P site, the bound tRNA interacts with rRNA as well as with r-proteins. Similarly, a group of several proteins and the 23S rRNA are involved in creating the peptidyl transferase site. The catalytic activity of this site is exercised by the RNA. The site has been localized on the central protuberance by the binding of puromycin.

A nascent protein debouches through the ribosome, away from the active sites, into the region in which ribosomes may be attached to membranes (see 8 Protein localization). A polypeptide chain emerges from the ribosome through an exit channel, which leads from the peptidyl transferase site to the surface of the 50S subunit. It emerges ~15 Å away from the peptidyl transferase site. It probably extends through the ribosome as an unfolded polypeptide chain until it leaves the exit domain, when it is free to start folding.


Internal Ribosome Entry Sites Provide Opportunities for Translational Control

Although approximately 90% of eukaryotic mRNAs are translated beginning with the first AUG downstream from the 5ʹ cap, certain AUGs can be skipped over during the scanning process. Cells can initiate translation at positions distant from the 5ʹ end of the mRNA, using a specialized type of RNA sequence called an internal ribosome entry site (IRES). In some cases, two distinct protein-coding sequences are carried in tandem on the same eukaryotic mRNA; translation of the first occurs by the usual scanning mechanism, and translation of the second occurs through an IRES. IRESs are typically several hundred nucleotides in length and fold into specific structures that bind many, but not all, of the same proteins that are used to initiate normal 5ʹ cap-dependent translation. 


Two mechanisms of translation initiation.
(A) The normal, capdependent mechanism requires a set of initiation factors whose assembly on the mRNA is stimulated by the presence of a 5ʹ cap and a poly-A tail .
(B) The IRES-dependent mechanism, seen mainly in viruses, requires only a subset of the normal translation initiating factors, and these assemble directly on the folded IRES

In fact, different IRESs require different subsets of initiation factors. However, all of them bypass the need for a 5ʹ cap structure and the translation initiation factor that recognizes it, eIF4E.

Initiation of Protein Synthesis in Bacteria
The ribosome is composed of a large and a small subunit, which are assembled on the translation initiation region (TIR) of the mRNA during the initiation phase of translation.

My comment "Know-how", foreknowledge, and goal orientedness is necessary for this machine-like process to occur. It has to be pre-programmed. Unguided, random events would never "know" that the small and large ribosomal subunits have to be assembled at the translation initiation region (TIR) for the initiation phase of translation to occur. There is simply no requirement for natural selection or any proposed mindless process to instantiate, program, and orchestrate such a complex precise process.  

The problem is that nature has too many options and without design couldn’t/wouldn't sort them all out. Natural mechanisms are too unspecific, chaotic, random, and without goal to determine any particular outcome. Natural processes could hypothetically form a machine-like assembly process, but also compatible with the formation of a wide variety of other molecular assemblages, most of which have no biological relevance or function. Random molecules have a total freedom of arrangements and chemical reactions. Yet it’s precisely that randomness that makes chemicals unable to account for specific mechanistic arrangements of remote possibility to happen. Chemicals without intent rather than resulting in structured functional ordered assembly of complex mechanistic structres give any non-functional outcome, and lead to doing anything. Yet when one of those things is a highly improbable specified event, design is the more case-adequate explanation. Occam's razor also boils down to an argument from ignorance: in the absence of better information, you use a heuristic to accept one hypothesis over the other.

In the following elongation phase, the mRNA is decoded as it slides through the ribosome and a polypeptide chain is synthesized. Elongation continues until the ribosome encounters a stop codon on the mRNA and the process enters the termination phase of protein synthesis. Newly synthesized protein is released from the ribosome. In the final ribosome recycling phase, the ribosomal subunits dissociate and the mRNA is released. Each phase is regulated by a number of different factors. Reviews of the phases are available.

My comment:  Regulation is ubiquitous in biology.

Biological regulation: controlling the system from within 06 August 2015
Regulation requires a distinctive architecture of functional relationships, and specifically the action of a dedicated subsystem whose activity is dynamically decoupled from that of the constitutive regime.
https://link.springer.com/article/10.1007/s10539-015-9497-8

All living organisms share several key characteristics or functions: order, sensitivity or response to the environment, reproduction, growth and development, regulation, homeostasis, and energy processing. When viewed together, these characteristics serve to define life.
https://courses.lumenlearning.com/wm-biology2/chapter/properties-of-life/

Although the main events of the translation process are universally conserved, major differences in the detailed mechanism of each phase exist. Bacterial translation involves relatively few factors, in contrast to the more complex process in eukaryotes (164). Here we focus on translation initiation in bacteria. Although parallels are drawn to the archaeal and eukaryotic systems where relevant, everything described throughout the rest of this review concerns the bacterial system unless otherwise stated. Archaeal and eukaryotic processes of translation initiation are reviewed elsewhere (7, 44, 177).

The Start Codon Is Chosen
A special tRNA, the initiator tRNA, is charged with methionine and binds to the AUG start codon


Initiator tRNA Carries N-Formyl-Methionine
(A) The structure of the initiator tRNA, fMet-tRNA, is unique. A CA base pair at the top of the acceptor stem is needed to allow formylation (violet). The initiator tRNA must enter the P-site directly (discussed later), which requires the three GC base pairs in the anticodon stem (blue).
(B) The initiator tRNA is first charged with unmodified methionine. Then a formyl group carried by the tetrahydrofolate cofactor is added to the
methionine.

In prokaryotes, chemically tagged methionine, N-formyl-methionine (fMet), is attached to the initiator tRNA, whereas in eukaryotes unmodified methionine is used. Consequently, all polypeptide chains begin with methionine, at least when first made. Sometimes the initial methionine (in eukaryotes), or N-formyl-methionine (in prokaryotes), is snipped off later, so mature proteins do not always begin with methionine. In bacteria, even when the fMet is not removed as a whole, the N-terminal formyl group is often removed leaving unmodified methionine at the N-terminus of the polypeptide chain. AUG codons also occur in the middle of messages and result in the incorporation of methionines in the middle of polypeptide chains. So how does the ribosome know which AUG codon to start with? Near the front (the 5' end) of the mRNA of prokaryotes is a special sequence, the ribosome-binding site (RBS), often called the Shine-Dalgarno or S-D sequence, after its two discoverers


Shine-Dalgarno Sequence of mRNA Binds to 16S rRNA
The Shine-Dalgarno sequence on the mRNA is recognized by base pairing with the anti-Shine-Dalgarno sequence on the 16S rRNA. The first AUG downstream of the S-D/anti-S-D site serves as the start codon.

The sequence complementary to this, the anti-Shine-Dalgarno sequence, is found close to the 3' end of the 16S rRNA. Consequently, the mRNA and the 16S rRNA of the ribosome bind together by base pairing between these two sequences. The start codon is the next AUG codon after the ribosome-binding site. Typically, there are about seven bases between the S-D sequence and the start codon. In some cases, the S-D sequence exactly matches the antiS-D sequence and the mRNAs are translated efficiently. In other cases, the match is poorer and translation is less efficient. (Note that eukaryotes do not use an S-D sequence to locate the start of translation; instead, they scan the mRNA starting from the 50-cap) Occasionally, coding sequences even start with GUG (normally encoding valine) instead of AUG. This leads to inefficient initiation and is mostly found for proteins required only in very low amounts, such as regulatory proteins, for example, LacI, the repressor of the lac operon. Note that when GUG acts as the start codon, the same initiator fMettRNA is used as when AUG is the start codon. Consequently, fMet is the first amino acid, even for proteins that start with a GUG codon. This is apparently due to the involvement of the initiation factors, especially IF3.

My comment: Let us suppose there was just a randomly polymerized RNA or DNA polypeptide chain, and the transcription and translation machinery to make proteins had not emerged yet. If the genetic code, and codon-words were not pre-established first, the sequence would be just gibberish, a random sequence. There would not be genetic words (codons), nor syntax being able to be recognized by the translation machinery nor tRNA's, and there would be no assignment of amino acids to make functional proteins.

Now lets suppose all the machinery was there, but just the Shine-Dalgarno sequence on the mRNA and its cognate anti-Shine-Dalgarno sequence on the 16S rRNA ( a ribosomal RNA member of the ribosome) were not extant.







Nobel Prize Winner Cites the "Ingeniously Designed" Architecture of the Ribosome

In 2009, Israeli structural biologist Ada Yonath shared the Nobel Prize in Chemistry for her work on the structure and function of the ribosome. More recently, she had this to say about the "ingeniously designed" architecture of the ribosome:

Ribosomes, the key players in the translation process, are universal ribozymes performing two main tasks: decoding genetic information and polymerizing amino acids. Hundreds of thousands of ribosomes operate in each living cell due to the constant degradation of proteins through programmed cell death, which is matched by simultaneous production of proteins. For example, quickly replicating cells, e.g. liver cells, may contain a few million ribosomes. Even bacterial cells may contain [up] to 100,000 ribosomes during their log period. Other constituents are the mRNA chains, produced by the transcription of the segments of the DNA that should be translated, which carry the genetic information to the ribosomes, and tRNA molecules bring the cognate amino acids to the ribosome. To increase efficiency, a large number of ribosomes act simultaneously as polymerases, synthesizing proteins by one-at-a-time addition of amino acids to a growing peptide chain, while translocating along the mRNA template, and producing proteins on a continuous basis at an incredible speed, namely up to 20 peptide bonds per second.

Ribosomes are giant assemblies composed of many different proteins (r-proteins) and long ribosomal RNA (rRNA) chains. Among these, the RNA moieties perform the two ribosomal main functions. The ratio of rRNA to r-proteins (~2:1) is maintained throughout evolution, except in mitochondrial ribosome (mitoribosome) in which ~half of the bacterial rRNA is replaced by r-proteins. Nevertheless, the active regions are almost fully conserved in all species. In all organisms ribosomes are built of two subunits, which associate to form the functionally active ribosomes. In prokaryotes, the small subunit, denoted as 30S, contains an RNA chain (16S) of ~1500 nucleotides and ~20 different proteins. The large subunit (50S in prokaryotes) has two RNA chains (23S and 5S RNA) of about 3000 nucleotides in total, and different >31 proteins. The available three dimensional structures of the bacterial ribosome and their subunits show that in each of the two subunits the ribosomal proteins are entangled within the complex rRNA conformation, thus maintaining a striking dynamic architecture that is ingeniously designed for their functions: precise decoding; substrate mediated peptide-bond formation and efficient polymerase activity.



Origin and Evolution of the Ribosome

Abstract :   likely, it is argued, were likely, likely order, may have, Finally, a highly speculative timeline of major events in ribosome history is presented and possible future directions discussed

When opponents of special creation asked about how x arised, they often make a quick web search, come up with the first search result which looks like a " serious " scientific paper, which explains how x  evolved, and post it as a answer. When asked to quote the relevant part of the paper, which convinced them evolution were the best answer, commonly they don't  answer, because they did not make the effort to analise carefully the proposed evidence. That shows nicely their confirmation bias. They determined already evolution must be true, since it fits their preconceived and wished world view, so all they do, is to try to fit everything they find into their naturalistic world view, without carefully looking if the evidence is compelling. Most scientific papers on evolution are perfect examples of how methodological naturalism works, and obliges specially historical sciences to wear blinkers.  Since evolution is the only naturalistic possible explanation for the biodiversity on earth, evolution is supposed to be the answer right from the beginning, rather to start with a agnostic standpoint , and after careful examination, permitting the evidence to lead wherever it is, and  propose evolution as the best explanation if that is the outcome that fits best. These pappers start with evolution, end with evolution, and in the middle is a not rarely high concentration of   guess work, ad hoc explanations , and fairy tale stories.

The modern ribosome was largely formed at the time of the last common ancestor, LUCA. Hence its earliest origins likely the guesswork begins lie in the RNA world. Central to its development were RNAs that spawned the modern tRNAs and a symmetrical region deep within the large ribosomal RNA, (rRNA), where the peptidyl transferase reaction occurs. To understand pre-LUCA developments, it is argued that events that are coupled in time are especially useful if one can infer a likely order in which they occurred. Using such timing events, the relative age of various proteins and individual regions within the large rRNA are inferred. An examination of the properties of modern ribosomes strongly suggests that the initial peptides made by the primitive ribosomes were likely enriched for l-amino acids, but did not completely exclude d-amino acids. This has implications for the nature of peptides made by the first ribosomes. From the perspective of ribosome origins, the immediate question regarding coding is when did it arise rather than how did the assignments evolve. The modern ribosome is very dynamic with tRNAs moving in and out and the mRNA moving relative to the ribosome. These movements may have become possible as a result of the addition of a template to hold the tRNAs. That template would subsequently become the mRNA, thereby allowing the evolution of the code and making an RNA genome useful. Finally, a highly speculative timeline of major events in ribosome history is presented and possible future directions discussed.

and as final not of the paper :
In the end, no matter how complete a picture is developed of ribosomal development over time it will be hypothetical. The ultimate issue will be to prove at least the major parts of it. Thus, laboratory reconstructions will be needed. However, there would be limited value in resurrecting the complete ribosome of LUCA, because it was in effect a modern ribosome itself.

http://cshperspectives.net/content/2/9/a003483.full

http://darwins-god.blogspot.com.br/2010/12/miracle-of-ribosome-assembly-evolution.html

New research is uncovering the details of how the cell’s protein factory—the ribosome—is constructed. The ribosome translates messenger RNA molecules—edited copies of DNA protein-coding genes—into a string of amino acids, according to the genetic code. The ribosome has two major components (one smaller and one larger), each made up of both RNA and protein molecules, and is constructed via a complex sequence of events.

The RNA components of the ribosome are copies of DNA genes. These ribosomal RNA molecules—known as rRNAs—are initially in a raw copy of DNA which eventually is edited. For instance, one of these copies of DNA may contain many rRNAs, separated by spacer segments. The spacer segments need to be removed, leaving the individual rRNAs ready for assembly.

One of the findings of the new research is that in the early stages of assembly, one of the DNA copies folds up such that a spacer segment binds to one of the rRNAs. In particular, the spacer binds to the special segment of the rRNA that reads the messenger RNA molecules, in the final, assembled ribosome. When the spacer is removed, the rRNA switches to its correct shape, for function in the ribosome. One implication of this finding is that ribosome construction can be regulated by this switch. Remove the spacer and ribosome construction proceeds. Leave the spacer, and ribosome construction halts. As the researchers concluded:

our data show that the intrinsic ability of RNA to form stable structural switches is exploited to order and regulate RNA-dependent biological processes.


RNA tends to fold up into a variety of shapes. In this case, as lead researcher Katrin Karbstein suggests, RNA folding properties are part of the design:

Perhaps, nature has found a way to exploit RNA’s Achilles’ heel—its propensity to form alternative structures … Nature might be using this to stall important biological processes and allow for quality control and regulation.

But of course this mechanism brings yet more complexity:

What is interesting is that as the organism becomes more complex, the number of cleavages needed increases. This may make the process more accurate and that may be an evolutionary advantage, but even in bacteria this cutting is not done in a simple way. We still don’t know exactly why that is.


Karbstein suggests that the strictly ordered cutting and pasting steps in ribosome assembly are introduced to produce singularly perfect intermediates. As she explains:

Ribosomes make mistakes rarely, on the order of one in 10,000 amino acid changes. A lot of this accuracy depends on conversations between different parts of the ribosomes, so if the structure of the RNA isn’t correct, these conversations can’t happen. And that means more mistakes, and that’s not good because it can lead to any number of disease states.


Ribosomes don’t just happen. They are not easily assembled and the evolution of this choreography calls for several just-so heroics. Yes, this fine-tuned set of mechanisms makes for fantastic regulation of the cellular protein making factory, but it means that evolution must have gone through a stage where life didn’t work. For the spacer segment that binds to the rRNA is a show-stopper. It would be selected against instantly.

The only way to resolve this problem is to have the spacer removal mechanism already in place, before the spacer sequence itself evolved. As usual, evolutionists would need to rely on the needed mechanism just happening to serve some other useful purpose, and when the spacer sequence happened to arise for no reason, the removal mechanism found new work for itself. In other words, mutations arose that caused the DNA copy to fold, rendering ribosome synthesis—and life itself—impossible. But as luck would have it, there just happened to be the right molecular machine lying around that removed the problematic segment at just the right time and place. Not only was the fatal flaw obviated, but a brilliant new means of regulation invented. Amazing. Over time, further mutations happened to refine its actions and today we have the fine-tuned ribosome assembly process.

There you have it—evolution’s just-add-water version of science. For the umpteenth time evolution becomes a charade. Behind the scenes, in deep-time where no one can see it working, evolution once again performs miracle after miracle.

An RNA conformational switch regulates pre-18S rRNA cleavage.

Abstract
To produce mature ribosomal RNAs (rRNAs), polycistronic rRNA transcripts are cleaved in an ordered series of events. We have uncovered the molecular basis for the ordering of two essential cleavage steps at the 3'-end of 18S rRNA. Using in vitro and in vivo structure probing, RNA binding and cleavage experiments, and yeast genetics, we demonstrate that a conserved RNA sequence in the spacer region between the 18S and 5.8S rRNAs base-pairs with the decoding site of 18S rRNA in early assembly intermediates. Nucleolar cleavage at site A(2) excises this sequence element, leading to a conformational switch in pre-18S rRNA, by which the ribosomal decoding site is formed. This conformational switch positions the nuclease Nob1 for cytoplasmic cleavage at the 3'-end of 18S rRNA and is required for the final maturation step of 18S rRNA in vivo and in vitro. More generally, our data show that the intrinsic ability of RNA to form stable structural switches is exploited to order and regulate RNA-dependent biological processes.

But the Ribosome is fascinating, and worth a closer look about its amazing properites and functions :

http://www.hopkinsmedicine.org/news/media/releases/Lost_In_Translation

The Ribosome: Perfectionist Protein-Maker Trashes Error

The enzyme machine that translates a cell's DNA code into the proteins of life is nothing if not an editorial perfectionist

Johns Hopkins researchers, reporting in the journal Nature January 7, have discovered a new "proofreading step" during which the suite of translational tools called the ribosome recognizes errors, just after making them, and definitively responds by hitting its version of a "delete" button.

It turns out, the Johns Hopkins researchers say, that the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products which, as workhorses of the cell, carry out the very business of life.

and it's this compounding of errors that leads to the partially finished protein being tossed into the cellular trash," she adds.

To their further surprise, the ribosome lets go of error-laden proteins 10,000 times faster than it would normally release error-free proteins, a rate of destruction that Green says is "shocking" and reveals just how much of a stickler the ribosome is about high-fidelity protein synthesis. "The cell is a wasteful system in that it makes something and then says, forget it, throw it out,"

That looks all ingeniously designed....... :smile: :smile: :thumbup:


Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis

Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm.

Ribosome biogenesis is a fundamental multistep process that, in eukaryotes, takes place largely within the nucleolus.Late steps in both 40S and 60S ribosomal subunit (r-subunit) synthesis occur in the nucleoplasm and after nuclear export of precursor particles in the cytoplasm . Ribosome synthesis is evolutionarily conserved throughout eukaryotes , and so far most of our understanding of this process has been obtained from studies with Saccharomyces cerevisiae . In the yeast nucleolus, three of the four rRNA (18S, 5.8S and 25S) are transcribed as a single large primary transcript by RNA polymerase I and processed to the first detectable rRNA precursor (pre-rRNA), the so-called 35S pre-rRNA. The fourth rRNA (5S) is independently transcribed as a pre-rRNA (pre-5S) by RNA polymerase III. In the 35S pre-rRNA, the mature rRNA sequences are separated by two internal transcribed spacers (ITS1 and ITS2) and flanked by two external transcribed spacers (5′ ETS and 3′ ETS), which must be precisely and efficiently processed to ensure correct formation of mature rRNAs . Maturation of rRNAs is a well-defined pathway  and involves numerous trans-acting factors that are required for the processing and covalent rRNA modification reactions, such as small nucleolar RNA–protein (snoRNP) complexes, endonucleases and exonucleases, and different base methylases . Concomitantly to rRNA maturation, the pre-rRNAs assemble in an ordered manner with the 79 ribosomal proteins (r-proteins) and a large number of trans-acting factors that are generally referred to as r-subunit assembly factors  (for examples of trans-acting factors see http://www.medecine.unige.ch/~linder/proteins.html). The process of r-subunit assembly is still poorly understood. An outline of this process was provided by sucrose density gradient analyses in the 1970s, which identified 90S, 66S and 43S pre-ribosomal particles . Recent advances employing proteomic approaches have revealed several distinct, successive pre-ribosomal particles and refined the model for the maturation of both 40S and 60S r-subunits [for a review . These proteomic approaches have also led to the identification of novel non-ribosomal proteins, increasing the number of trans-acting factors involved in ribosome biogenesis to over 180. Evidence towards an understanding of the function of many of these trans-acting factors has been obtained by using a complete repertoire of techniques, thus, addressing their temporal association with pre-ribosomal particles and revealing the pre-rRNA processing and nucleocytoplasmic export defects caused by their mutational inactivation or depletion .

http://www.ncbi.nlm.nih.gov/pubmed/21529161
Ribosome assembly needs  the contributions of several  assembly cofactors , including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.


http://rnajournal.cshlp.org/content/10/12/1833.full

recent crystallographic studies have revealed the ribosome to be a structure of unprecedented complexity

Most importantly, they cannot have evolved initially to make functional proteins, in the modern sense, because of the vanishingly small probability that the first attempts at polypeptide synthesis by a primitive translational apparatus could yield a protein with any useful enzymatic activity (Woese 1967).

That is to say, the RNA world could not have anticipated that the evolution of a macromolecular machine with the complexity of the ribosome would in turn eventually lead to the evolution of long polypeptide chains of specific sequence that fold into stable, three-dimensional structures with desirable biological functions. Clearly, until the first active proteins emerged, no selective advantage would exist for evolution of a translational machinery, if its only purpose was to synthesize functional proteins.

Yet, we know it evolved, because here it is ( funny, as if there were no other options, like special creation...... )

More likely, protein synthesis initially arose not to create fully functional enzyme-like proteins, but for some other purpose. ( how do they know ? )

Specification of amino acids by RNA sequences most likely emerged later, requiring coevolution of the ribosome and its tRNAs (Noller 1993; ( further guesswork... nice ! )


The Ribosome
http://www.evolutionnews.org/2012/03/study_questions057501.html

Molecular basis for protection of ribosomal protein L4 from cellular degradation
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296656/

Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function tofacilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery.

Assembly and functionality of the ribosome with tethered subunits

The ribosome as a missing link in the evolution of life 1

https://reasonandscience.catsboard.com/t1661-translation-through-ribosomes-amazing-nano-machines#5929


My comment:The title is already deceptive. There was no evolution prior to DNA replication. Why do the authors try to insert the mechanism of evolutionary change into a scenario pre-life, where it does not belong?

Hypothesize that ribosome was self-replicating intermediate between compositional or RNA-world and cellular life.

My comment: There is no explanation of what function the Ribosome would have without all the other parts required to make proteins.

rRNA contains genetic information encoding self-replication machinery: all 20 tRNAs and active sites of key ribosomal proteins.

My comment: the make of transfer RNA's ( tRNA's) requires ultracomplex molecular machinery and in a production line fashion, where several proteins and ribozymes work in a coordinated fashion to produce the twenty tRNA's used in life:

Transfer RNA, and its biogenesis, best explained through design
https://reasonandscience.catsboard.com/t2070-transfer-rna-and-its-biogenesis

tRNA's are very specific molecules, and the " made of " follows several steps, requiring a significant number of proteins and enzymes, which are often made of several subunits and ainded by essential co-factors and metals.

Statistical analyses demonstrate rRNA-encodings are very unlikely to have occurred by chance.

My comment: EXACTLY. But that is precisely the ONLY possible mechanism prior to DNA replication. 

The possible mechanisms to explain the origin of life
https://reasonandscience.catsboard.com/t2515-the-possible-mechanisms-to-explain-the-origin-of-life

Natural selection is not a possible mechanism to explain the origin of life, since evolution depends on DNA replication

When we consider how life might have arisen from nonliving matter, we must take into account the properties of the young Earth’s atmosphere, oceans, and climate, all of which were very different than they are today. Biologists postulate that complex biological molecules first arose through the random physical association of chemicals in that environment.
LIFE The Science of Biology, TENTH EDITION, page 3

Neither Evolution nor physical necessity are a driving force prior dna replication. The only two alternatives are either a) creation by an intelligent agency, or b) Random, unguided, undirected natural events by a lucky "accident".

Koonin, the logic of chance, page 266
Evolution by natural selection and drift can begin only after replication with sufficient fidelity is established. Even at that stage, the evolution of translation remains highly problematic. The emergence of the first replicator system, which represented the “Darwinian breakthrough,” was inevitably preceded by a succession of complex, difficult steps for which biological evolutionary mechanisms were not accessible . The synthesis of nucleotides and (at least) moderate-sized polynucleotides could not have evolved biologically and must have emerged abiogenically—that is, effectively by chance abetted by chemical selection, such as the preferential survival of stable RNA species. Translation is thought to have evolved later via an ad hoc selective process.  Did you read this ???!! An ad-hoc process ??

Suggest that DNA and cells evolved to protect and optimize pre-existing ribosome functions.

My comment: There were no self-replicating cells without the Ribosome fully setup and operating....

Among the molecules that probably arose very early in the biological systems, the large subunit of the ribosomal RNA that composes the ribosomes in cellular organisms are considered as a possible turning point in molecular evolution (Fox, 2010). Even more specifically, a part of this molecule called the Peptidyl Transferase Center (PTC) is considered by some as having an essential role in evolution, since this catalytic ability of getting
together aminoacids is crucial for protein synthesis and thus, for a first transition fromaRNAworld to a Ribonucleoprotein world, as seen in modern organisms 2

A “selfish ribosome” as the origin of cellular life
To imagine a self-replicating ribosome is therefore to imagine that rRNA encodes “genes” for, 1) the rRNA scaffold itself, 2) the mRNAs required to encode the 50 or so ribosomal proteins, 3) the tRNAs that are required to “read” the mRNA, and 4) the protein enzymes required to put the amino acids on the tRNAs in preparation for protein synthesis. Since the current dogma is that rRNA encodes no genes whatsoever, let alone a complete set of tRNAs, this suggestion required a huge imaginative leap.
https://atlasofscience.org/a-selfish-ribosome-as-the-origin-of-cellular-life/


A . Earliest Beginnings: RNA World

1. Initial Formation of Peptidyl Transferase Center in RNA World
A. Emergence of RNAs that can aminoacylate RNAs -leads to small charged
RNAs (minihelices)
B. Emergence of RNAs that can catalyze peptide bond formation between minihelices.
2. Beginnings of coherent SOS subunit
Portions of Domain V of 23S rRNA likely present. Addition of more RNA or
essentially random peptides might have decreased hydrolysis reaction and increase
activity by protecting the core reaction.
3. Extension of tRNA to two domains;
Second tRNA domain allows templating, which significantly increases reaction rate.
Characteristic conformational changes associated with translocation are present.
4. Beginnings of coding
It is now possible to store information. This makes it useful to have a genome (RNA)
and hence primitive polymerases might offer a significant selective advantage to
progenotes that have them.
B. Beginnings of Transition to Protein World: Late RNA World
5. Ancestors of core proteins such as L3 and L4 are present.
Emergence of defined sequence peptides means RNA World will soon end.
6. Initial creation of 30S particle
Further protection of the reaction machinery is possible by stabilizing template.
Bridges between subunits were probably initially only RNA. Portions of Domain IV
of 23S rRNA that interact with 30S subunit are likely to be present.
C. Early Protein World: Major Refinements Increase Speed and Accuracy
7. EF-Tu ancestor
Improved control of tRNA access to machinery
8. Addition of 5S rRNA complex
Further development of SOS subunit underway. Many new proteins present such as LS
and LI 8 that are associated with SS rRNA incorporation
9. GTPase Reaction Center Formed
The emerging protein world allows the development of the translocation machinery.
LI 0 now present. At least portions of 23S rRNA Domain II are present.

The ribosome as a missing link in the evolution of life
We hypothesize that ribosome was self-replicating intermediate between compositional or RNA-world and cellular life. We suggest that the ribosome may represent one important missing link between compositional(or metabolism-first), RNA-world(or genes-first)and cellular (last universal common ancestor) approaches to the evolution of cells. Our results clearly favor the hypothesis that the ribosome could have been a self-organizing, self-replicating pre-cellular entity. A self-replicating ribosomal entity would provide a logical intermediary between selfreplicating RNAs or compositionally-organized aggregates of molecules and highly organized, cell-encapsulated genomes. “Selfish” ribosomes, in short, provide one potential intermediary in the process of evolution from the first macromolecules to hyperstructures and finally cells.
https://www.sciencedirect.com/science/article/pii/S0022519314006778

Ribosomes and the tree of life March 7, 2016
In 1969, Associate Professor of Microbiology Carl Woese wrote to Nobel Laureate Francis Crick seeking moral support for an investigation of the distant past. Woese wrote:
…I would be grateful… for any backing (largely moral) you could give me… if we are ever to unravel the course of events leading to the evolution of the prokaryotic (i.e., simplest) cells… this can be done by using the cell’s “internal fossil record”. The obvious choice of molecules here lies in the components of the translation apparatus. What more ancient lineage is there?
http://serious-science.org/ribosome-and-the-origin-of-life-5814#:~:text=During%20or%20near%20the%20origin,Central%20Dogma%20of%20Molecular%20Biology.&text=The%20ribosome%20is%20a%20lens,molecules%2C%20from%20before%20protein%20synthesis.

EVOLUTION OF LIFE'S OPERATING SYSTEM REVEALED IN DETAIL Jul 7, 2014
“The translation system is the operating system of life,” Williams said. “At its core the ribosome is the same everywhere. The ribosome is universal biology.”
https://www.astrobio.net/also-in-news/evolution-lifes-operating-system-revealed-detail/

Evolution of the ribosome at atomic resolution
Building Up the Peptidyl Tranferase Center: The PTC is an essential component of the ribosome, responsible for peptide bond formation. The PTC is thought to predate coded protein and is believed to be among the oldest polymeric elements of biological systems.
https://www.pnas.org/content/111/28/10251

1. https://www.sciencedirect.com/science/article/pii/S0022519314006778
2. http://sci-hub.tw/https://www.cambridge.org/core/journals/international-journal-of-astrobiology/article/buds-of-the-tree-the-highway-to-the-last-universal-common-ancestor/ED26AA7787BA5A152090913CC7C20067

The Ribosome is like a 3D printer

From Darwin to Eden: A Tour of Science and Religion based on the Philosophy ... By William B. Collier



CASPAR HENDERSON A NEW MAP OF WONDERS: 
The first is the ribosome: a tiny ‘factory’ that makes all the proteins essential for life. (There are about twenty-five thousand different kinds of protein in a human body. Most individual protein molecules last only a few days so they need to be steadily replaced.) Ribosomes are so fundamental that it is hard to see how cells as we know them could ever have come to be without them. They are found in every cell of everything alive, and have the same essential structure in all of them. Their active sites and central cores are built of entirely of RNA.  Compared with molecules such as water and simple sugars, ribosomes are enormous, consisting of about a million atoms; but compared with cells they are tiny, and a typical human cell can contain many millions of them. Each ribosome, which consists of large and small subunits like parts of a robotic press, reads information conveyed to it from DNA in the cell nucleus by messenger RNA rather as if it were reading brail, and uses the information to select and then stamp together amino acids so that they form new proteins. It does this at the rate of about forty per second, and with an error rate of less than one in ten thousand – far better than humans achieve in high-quality manufacturing. All in a space just twenty to thirty nanometers across. The physicist Neil Gershenfeld calls ribosomes the original digital fabricators, 4 billion years ahead of 3D printers, and vastly more capable and reliable. With a 3D printer, the design is determined by a computer program, which is digital, but the material with which it works, such as a resin, has no self-organizing properties: it is just kind of smooshy. In the case of a ribosome, however, the twenty amino acids it ‘prints’ with come in regular and repeating shapes – a fair analogy is Lego bricks – and this makes fabrication repeatable and precise even as a very large number of configurations is possible. To an extent, the ‘code’ is also in the material, because the shape of the parts directs them to configure in a limited number of ways. The ribosome manufactures all the proteins essential to life in every living thing, and likely predates life as we know it.

Hasan DeMirci refers to ribosomes – tiny molecular machines made up of tangles of RNA and proteins clumped together and intricately wired like ramen noodles in soup – as “the 3D printers of the human body.” The ribosomes synthesize proteins using the genetic information contained in DNA, “building our bodies from the ground up.” 3

Polyribosomes Are Molecular 3D Nanoprinters That Orchestrate the Assembly of Vault Particles
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245718/
Structural and functional evidence points to a model of vault assembly whereby the polyribosome acts like a 3D nanoprinter to direct the ordered translation and assembly of the multi-subunit vault homopolymer, a process which we refer to as polyribosome templating. Successive addition of growing MVP dimers, layer-by-layer like in a 3D printing process, continues until the 78-mer of MVPs is reached and completes the entire vault structure at the 3′ end of a polyribosome. In a time in which efficient 3D manufacturing is predicted to have a revolutionary effect on mankind, nature unveils that it has already been using this technique for millions of years. Vaults are very large ribonucleoprotein particles found widely in eukaryotes. Our discovery of the unique assembly mechanism of the vault particle reveals an unforeseen function of the polyribosome as a very sophisticated cellular 3D nanoprinter.  4

1. https://www.embopress.org/doi/full/10.1093/emboj/cdg017
2. https://books.google.com.br/books?id=2tbaDwAAQBAJ&pg=PA118&lpg=PA118&dq=ribosome+is+a+3d+Printer+machine&source=bl&ots=xhNX4hieOA&sig=ACfU3U2ek0b6YYyAgyNQXJtrS5URizju_w&hl=en&sa=X&ved=2ahUKEwiyx56Cs8LuAhUtuVkKHSPVAjsQ6AEwF3oECFgQAg#v=onepage&q&f=false
3. https://www6.slac.stanford.edu/news/2018-08-06-catching-dance-antibiotics-and-ribosomes-room-temperature.aspx#:~:text=Hasan%20DeMirci%20refers%20to%20ribosomes,building%20our%20bodies%20from%20the
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245718/

Termination of translation: interplay of mRNA, rRNAs and release factors?
Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain release factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.

Termination of translation exemplifies in a striking way how informational and chemical aspects of biology can be integrated fundamentally. Termination signals of polypeptide synthesis are encoded in the genome and transcribed to mRNA in the form of three different base triplets referred to as termination, stop or nonsense codons. When a stop codon has been translocated into the ribosomal A‐site by the action of elongation factor EF‐G or eEF2, it is decoded at the small ribosomal subunit. However, the chemical reaction that is triggered by a stop signal, the cleavage of the ester bond between the peptidyl and tRNA moieties of the peptidyl‐tRNA, occurs within the large ribosomal subunit at the peptidyl transferase centre (PTC) of the ribosome. How a stop signal can be transduced from the small to the large ribosomal subunit and trigger hydrolysis of peptidyl‐tRNA remains unknown.

Class‐1 polypeptide release factors decode stop signals in mRNA
Decoding of the stop signals in mRNAs could, in principle, be implemented by 

(i) the ribosome itself; 
(ii) external factors that associate with the ribosome in response to a stop signal and then dissociate after completed action; and 
(iii) the combined action of the ribosome and external factors. 

It was suggested that either the small or the large rRNA can interact directly with stop codons in mRNA. This version of the first hypothesis is supported by data from several experiments. First, mutations in the small and large rRNAs strongly affect translational termination. Secondly, the stop codon is in a close contact with rRNA within the ribosome. Thirdly, two segments of prokaryotic 23S rRNA (helices 69 and 89) can be folded into a tRNA‐like shape with anticodon‐like loops complementary to stop codons. However, this in silico‐derived hypothesis lacks support from experiments.

It is known that one or several external factors, called class‐1 polypeptide release factors (RFs), associate(s) transiently with the ribosomal A‐site. In bacteria, there are two RFs, the proteins RF1 and RF2, which recognize UAA/UAG and UAA/UGA, respectively. In eukaryotes, archaea and mitochondria, in contrast, only a single protein (eRF1, aRF1 and mtRF1, respectively) exists. In Arabidopsis thaliana and in the ciliate Euplotes, eRF1 is present as two homologous molecular species. It remains unknown whether these eRF1 homologues respond to the same or different stop codons. The fact that external factors exist and terminate protein synthesis in a codon‐specific way does not in itself tell us whether they recognize stop codons through direct interactions (second hypothesis) or indirectly, via rRNA (third hypothesis).

Ribosome biogenesis

https://www.youtube.com/watch?v=2guXr5c4rHc
There are factors that facilitate the assembly. It is initially made in a much longer sequence and then cut into the pieces themselves being precursors which are subsequently cleaved down to the correct size the RNA is modified both on the sugars and modified into the bases at a number of places

Question: How did random prebiotic forces "discover" where to cut and modify the RNA strands to the correct size ?

https://en.wikipedia.org/wiki/Ribosome_biogenesis
Ribosome biogenesis is the process of making ribosomes. In prokaryotes, this process takes place in the cytoplasm with the transcription of many ribosome gene operons. In eukaryotes, it takes place both in the cytoplasm and in the nucleolus. It involves the coordinated function of over 200 proteins in the synthesis and processing of the three prokaryotic or four eukaryotic rRNAs, as well as assembly of those rRNAs with the ribosomal proteins. Most of the ribosomal proteins fall into various energy-consuming enzyme families including ATP-dependent RNA helicases, AAA-ATPases, GTPases, and kinases. About 60% of a cell's energy is spent on ribosome production and maintenance.

Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae 1 November 2013
https://academic.oup.com/genetics/article/195/3/643/5935478?login=true
Assembly of the ∼5500 nt of RNA and 79 r-proteins into ribosomes requires 76 different small nucleolar RNAs and >200 different assembly factors. Not only is this construction project expensive, but also it must be completed many times and in a hurry! More than 2000 ribosomes are assembled each minute in a rapidly growing yeast cell. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.

RIBOSOMES are the cellular machines that translate the genetic code in mRNA and catalyze protein synthesis in all organisms. These ancient, complex nanomachines consist of two ribonucleoprotein subunits. The small, 40S subunit (SSU) in yeast contains one ribosomal (r)RNA [18S, 1800 nucleotides (nt) long] and 33 different ribosomal proteins (r-proteins) while the large, 60S subunit (LSU) includes three rRNAs (5S, 121 nt; 5.8S, 158 nt; and 25S, 3396 nt) plus 46 r-proteins. The SSU serves as the decoding center to bring mRNA and aminoacylated transfer (t)RNAs together. The LSU is where the peptidyltransferase reaction occurs to catalyze peptide bond formation.

Ribosome assembly is a major undertaking for cells, requiring a significant fraction of the resources devoted to macromolecular synthesis and trafficking. All three RNA polymerases participate. RNA polymerases I and III transcribe the rRNAs. The messenger (m)RNAs encoding r-proteins and ribosome assembly factors comprise at least 60% of the transcripts produced by RNA polymerase II and ultimately translated by ribosomes. More than one-half of the introns removed by the yeast splicing machinery are those present in r-protein pre-mRNAs. A major class of nuclear import and export cargo includes r-proteins and assembly factors entering the nucleus and pre-ribosomes exiting the nucleus. Assembly of the ∼5500 nt of RNA and 79 r-proteins into ribosomes requires 76 different small nucleolar RNAs and >200 different assembly factors. Not only is this construction project expensive, but also it must be completed many times and in a hurry! More than 2000 ribosomes are assembled each minute in a rapidly growing yeast cell.

Ribosome construction also is subject to stringent inspection; elaborate mechanisms monitor nascent ribosomes for correct assembly. This quality control is coupled to nuclear export of pre-rRNPs and remarkably includes testing “teenage” cytoplasmic ribosomes for proper assembly, immediately before the last steps of subunit construction.

Because production of ribosomes is so closely tied to the growth and proliferation of cells, dysregulation of ribosome assembly has profound consequences on the health of organisms. Complete loss-of-function mutations in most assembly factors and r-proteins are lethal in yeast and embryonic lethal in higher organisms. Perhaps more widespread is partial loss-of-function or haploinsufficiency of these ribosomal molecules, which in humans can present as a variety of maladies, including short stature, mental retardation, joint abnormalities, bone marrow failure, craniofacial dysmorphology, and predisposition to cancer. Pathways and participants in ribosome biogenesis in eukaryotes are proving to be largely conserved. Thus a complete understanding of ribosome assembly in yeast will enable more rapid understanding of the molecular basis of human disease caused by ribosomopathies.

The structures show that RNA is present in the core of each subunit, with r-proteins embedded on the surface and sometimes protruding into the rRNA core. The 18S rRNA sequences form four phylogenetically conserved secondary structural domains: the 5′, central, 3′ major, and 3′ minor domains (Figure 1A, left)


(A) Secondary structure of S. cerevisiae 18S, 25S, and 5.8S rRNAs. Left, the four domains of 18S rRNA secondary structure are indicated in different colors. Right, 25S rRNA contains six domains (I–VI) of secondary structure. The 5.8S rRNA (black) base pairs with domain I (adapted from www.rna.ccbb.utexas.edu). These secondary structures are phylogenetically conserved throughout all kingdoms, although eukaryotic rRNAs contain expansion segments not found in prokaryotic or archaeal rRNAs. (B) Tertiary structures of 18S rRNA (left) and 25S plus 5.8S rRNAs (right), from the crystal structure of yeast ribosomes (Ben-Shem et al. 2011).

These fold into tertiary structures (Figure 1B, left), which, together with r-proteins, form the body, shoulder, platform, head, and beak structures observed in SSUs (Figure 2A). The 5.8S and 25S rRNAs in the LSU fold into six conserved domains of secondary structure (I–VI), including base pairing between 5.8S and 25S rRNAs (Figure 1A, right). These six secondary domains then fold into tertiary structures found in mature 60S subunits (Figure 1B, right). Clearly discernible features of the LSU include the central protuberance (containing 5S rRNA), the L1 stalk, and the acidic stalk (Figure 2B). Comparison of these eukaryotic ribosome structures with those of bacterial and archaeal ribosomes reveals a universally conserved structural core that contains the sites for ribosome functions: the peptidyltransferase center, the polypeptide exit tunnel, and the GTPase binding site in LSUs and the tRNA-binding sites (A, P, and E), the decoding center, and mRNA entry and exit sites in SSUs. Features specific to eukaryotes include rRNA expansion segments and long amino- or carboxy-terminal extensions of many r-proteins, located mostly on the solvent-exposed surface of the subunits.

Ribosomes Are Made in the Cell Nucleolus
Ribosomes are made in a non-membrane-bound subcompartment of the cell nucleus termed the nucleolus. The single Saccharomyces cerevisiae nucleolus is formed around the ∼150 tandem repeats of the rDNA transcription unit found on chromosome XII (Figure 3)


Crystal structure at 3.0-A resolution of S. cerevisiae 40S and 60S ribosomal subunits. (A) Views of the solvent-exposed surface (left) and subunit interface (right) of the 60S subunit. CP, central protuberance. (B) The solvent-exposed surface (left) and the subunit interface (right) of the 40S subunit. rRNA is represented in gray and r-proteins are in red. 

and is defined by the act of rRNA transcription. Its crescent shape is detectable via immunostaining of known nucleolar proteins .


Organization of the rDNA locus in S. cerevisiae. The rDNA repeats (150–200) are located on chromosome 12. A single repeated unit is transcribed by RNA polymerase I (RNA pol I) to synthesize the 35S primary pre-rRNA transcript that will be processed to produce the mature 18S, 5.8S, and 25S rRNAs (arrow pointing right) and by RNA polymerase III (RNA pol III) to synthesize the 5S rRNA (arrow pointing left). NTS, nontranscribed spacer; ETS, external transcribed spacer; ITS, internal transcribed spacer.

RNA polymerase I is the specialized RNA polymerase that transcribes the pre-rRNA
Among the three RNA polymerases in eukaryotic cells, RNA polymerase I is the busiest. This enzyme transcribes the pre-rRNA that is processed to yield mature 18S, 5.8S, and 25S rRNAs. This represents 60% of the total cellular RNA transcription. In a single typical yeast cell generation, an astonishing 200,000 ribosomes are produced. To achieve this, RNA polymerase I maintains an elongation rate of 40–60 nt/sec. The ribosome biogenesis machinery consisting of pre-rRNA processing and preribosome assembly factors must keep up this demanding rate of pre-rRNA synthesis.

Transcription of the 35S primary transcript by RNA polymerase I occurs in the cell nucleolus. The initial 6.6-kb pre-rRNA includes RNAs destined for both the SSU (18S) and the LSU (5.8S and 25S) (Figure 3). The 35S pre-rRNA also bears RNA sequences that do not become part of the mature ribosome and are processed away: the 5′ external transcribed spacer (5′ETS), internal transcribed spacers 1 and 2 (ITS1 and ITS2), and part of the noncoding transcribed spacer 3′ to the 25S coding sequence (3′ETS). The third RNA that is part of the LSU, the 5S rRNA, is transcribed by RNA polymerase III in the opposite direction.

The S. cerevisiae RNA polymerase I holoenzyme has 14 subunits and is 590 kDa in size. Of the 14 subunits, all but Rpa34 and Rpa49 are shared or homologous with subunits in RNA polymerases II and III. A 12-Å cryo-EM structure of the complete RNA polymerase I and X-ray structures of subcomplexes reveal that the Rpa49/Rpa34 heterodimer retains features similar to those of TFIIF and TFIIE, further extending the similarities between RNA polymerase I and the other RNA polymerases. Interestingly, pioneering mass spectrometry has revealed potential subcomplexes of RNA polymerase I that may serve as building blocks for its assembly.

Four general transcription factors or transcription factor complexes aid in the recruitment of RNA polymerase I to the promoter. They include UAS-binding upstream activity factor (UAF) (composed of Rrn5, Rrn9, Rrn10, and histones H3 and H4), TATA-binding protein (TBP), core factor (CF) (composed of Rrn6, Rrn7, and Rrn11 proteins and analogous to SL1 in mammals), and Rrn3 (TIF1A in mammals). Interestingly, only ∼2% of RNA polymerase I is competent for transcription in yeast whole-cell extracts, and the initiation-competent RNA polymerase I is tightly associated with Rrn3 (Milkereit and Tschochner 1998). Furthermore, yeast Rrn7 and its human ortholog, TAF1B, play the role of TFIIB, functionally conserved among RNA polymerases, in preinitiation complex formation.

How is RNA polymerase I transcription regulated?
Classic experiments have demonstrated that ribosome biogenesis in yeast parallels growth rate. The rRNA synthesis step in ribosome biogenesis is regulated by transcription initiation and elongation and by the ratio of active to inactive rDNA repeats. During growth of S. cerevisiae possessing an unperturbed rDNA locus, the number of active genes decreases from log to stationary phase, indicating that the proportion of active genes can be modulated between growth conditions.

Measuring transcription by RNA polymerase I
There are several ways in which transcription of the rDNA can be assessed and quantified. Perhaps the most elegant is its direct visualization in chromatin spreads, using the electron microscope. First pioneered by Oscar Miller  using amphibian oocytes , the Beyer laboratory has recently used chromatin spreads to answer important questions about transcription and pre-rRNA processing in S. cerevisiae. Increasing lengths of rRNAs in the midst of its synthesis can be seen radiating from a strand of rDNA in chromatin spreads of the repeated units, with a dot of RNA polymerase I where each rRNA and rDNA strand meet (Figure 4).


Yeast chromatin spreads of nucleolar contents analyzed by electron microscopy. Transcription of the repeated rDNA units can be visualized as “Christmas trees”, where the trunk of the tree is the rDNA, the branches are the rRNA, and the ornaments are the knobs on the 5′ ends. The knob contains the SSU processome.

Counting the number of RNA polymerases in each transcription unit is one way of directly quantifying rRNA transcription. The Miller chromatin spreads are often referred to as “Christmas trees”, with the rDNA representing the tree trunk and the rRNA, the branches, and the nascent RNPs (seen as knobs on the 5′ ends) representing the ornaments. A second quantitative method to assess rDNA transcription is nuclear run-on assays, where incorporation of 32P[UTP] is measured after Sarkosyl is added to prevent reinitiation. A third method that is often used is the intensity of the 35S band, which is the primary transcript. In most cases, this does accurately echo what is seen with the other methods. However, this band results from the first pre-rRNA cleavage step at the 3′ end by Rnt1. Its intensity would thus result from the balance of rRNA synthesis, processing, and degradation. Since it is not the nascent transcript, but the product of the first processing step, there may be cases where its levels do not accurately reflect transcription.

Ribosome biogenesis is a very tightly regulated process, and it is closely linked to other cellular activities like growth and division.

Some have speculated that in the origin of life, ribosome biogenesis predates cells, and that genes and cells evolved to enhance the reproductive capacity of ribosomes.

Eukaryotic biogenesis
Ribosomal protein synthesis in eukaryotes is a major metabolic activity. It occurs, like most protein synthesis, in the cytoplasm just outside the nucleus. Individual ribosomal proteins are synthesized and imported into the nucleus through nuclear pores.

The DNA is transcribed, at a high speed, in the nucleolus, which contains all 45S rRNA genes. The only exception is the 5S rRNA which is transcribed outside the nucleolus. After transcription, the rRNAs associate with the ribosomal proteins, forming the two types of ribosomal subunits (large and small). These will later assemble in the cytosol to make a functioning ribosome.

Eukaryotic cells co-transcribe three of the mature rRNA species through a series of steps. The maturation process of the rRNAs and the process of recruiting the r-proteins happen in precursor ribosomal particles, sometimes called pre-ribosomes, and takes place in the nucleolus, nucleoplasm, and cytoplasm. The yeast, S. cerevisiae is the eukaryotic model organism for the study of ribosome biogenesis. Ribosome biogenesis starts in the nucleolus. There, the 18S, 5.8S, and 25S subunits of the rRNA are cotranscribed from ribosomal genes as a polycistronic transcript by RNA polymerase I, and is called 35S pre-RNA.

Transcription of polymerase I starts with a Pol I initiation complex that binds to the rDNA promoter. The formation of this complex requires the help of an upstream activating factor or UAF that associates with TATA-box binding protein and the core factor (CF). Together the two transcription factors allow the RNA pol I complex to bind with the polymerase I initiation factor, Rrn3. As the pol I transcript is produced, approximately 75 small nucleolar ribonucleoparticles (snoRNPs) facilitate the co-transcriptional covalent modifications of >100 rRNA residues. These snoRNPs control 2’-O-ribose methylation of nucleotides and also assist in the creation of pseudouridines. At the 5’ end of rRNA transcripts, small subunit ribosomal proteins (Rps) and non-ribosomal factors assemble with the pre-RNA transcripts to create ball-like knobs. These knobs are the first pre-ribosomal particles in the small (40S) ribosomal subunit pathway. The rRNA transcript is cleaved at the A2 site, and this separates the early 40S pre-ribosome from the remaining pre-rRNA that will combine with large subunit ribosomal proteins (Rpl) and other non-ribosomal factors to create the pre-60S ribosomal particles.

40S subunit
The transcriptional assembly of the 40S subunit precursor, sometimes referred to as the small subunit processome (SSU) or 90S particle happens in a hierarchical fashion – essentially a stepwise incorporation of the UTP-A, UTP-B, and UTP-C subcomplexes. These subcomplexes are made up of over 30 non-ribosomal protein factors, the U3 snoRNP particle, a few Rps proteins, and the 35S pre-rRNA. Their exact role, though has not been discovered. The composition of the pre-40S particle changes drastically once cleavage at the U3 snoRNPA dependent sites (sites A0, A1, and A2) are made. This cleavage event creates the 20S pre-rRNA and causes ribosomal factors to dissociate from the pre-40S particle. U3 is displaced from the nascent 40S by the helicase Dhr1. At this point in the ribosome biogenesis process, the 40S pre-ribosome already shows the “head” and “body” structures of the mature 40S subunit. The 40S pre-ribosome is transported out of the nucleolus and into the cytoplasm. The cytoplasmic 40S pre-ribosome now contains ribosomal proteins, the 20s rRNA and a few non-ribosomal factors. The final formation of the 40S subunit “beak” structure occurs after a phosphorylation and dephosphorylation event involving the Enp1-Ltv1-Rps3 complex and the kinase, Hrr25. Cleavage of the 20S pre-rRNA at the D-site creates the mature 18s rRNA. This cleavage event is dependent on several non-ribosomal factors such as Nob1, Rio1, Rio2, Tsr1 and Fap7.



Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo 6 February 2015
https://sci-hub.ren/10.1146/annurev-biochem-060614-033917
The proteome of cells is synthesized by ribosomes, complex ribonucleoprotein that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA co-transcriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth.

Ribosomes are complex ribonucleoprotein (RNP) machines that catalyze protein synthesis in all cells. Ribosomes consist of two subunits; the large subunit (LSU) is about twice the size of the small subunit (SSU). The SSU functions as the decoding center to bring together messenger RNAs(mRNAs) and aminoacyl–transferRNAs(tRNAs) to translate the genetic code. Coordinated conformational changes within the SSU also allow for translocation of the tRNA/mRNA pair through the ribosome. The eukaryotic SSU contains an 18S ribosomal RNA (rRNA) (1,800 nucleotides in yeast) and 33 different ribosomal proteins (r-proteins), which are organized into three distinct structural subdomains: the body, which contains the 5' domain of 18S rRNA; the platform, which contains the central domain; and the head, which contains the 3' major domain (Figure 1a,b).


Figure 1 Crystal structure of (a,b) the small subunit (SSU) and (c,d ) the large subunit (LSU) from Saccharomyces cerevisiae at 3.0-A˚ resolution. (a,c) The subunit interface of the SSU and LSU, respectively. (b,d) The
solvent-exposed surface of the SSU and LSU, respectively. Abbreviations: CP, central protuberance. GAC, GTPase-activation center. The crystal structure is adapted from Protein Data Bank codes 3U5B, 3U5C, 3U5D, and 3U5E.

The eukaryotic LSU, which houses the peptidyltransferase center (PTC) that catalyzes peptide bond formation, contains 5S rRNA (121 nucleotides in yeast), 5.8S rRNA (158 nucleotides in yeast), 25S–28S rRNA (25S rRNA; 3,396 nucleotides in yeast), and 46 (in yeast) or 47 (in human) r-proteins. The 25S rRNA is composed of six different rRNA domains, which are more intertwined with each other than are domains in the SSU. Thus, the LSU was originally described as largely monolithic, with only a few notable structural features, such as the central protuberance (CP) and the L1 and acidic stalks (Figure 1c,d ). However, we have now learned that the LSU is indeed partitioned into neighborhoods that assemble sequentially (see the section titled Assembly of 60S Ribosomal Subunits, below). These correspond at least in part to the previously defined secondary-structure domains. How these rRNAs and r-proteins associate with each other to form functional ribosomes has been a challenging and important problem investigated almost since the discovery of ribosomes. We now know that in eukaryotes ribosome biogenesis begins with the transcription of two precursor rRNAs (pre-rRNAs) in the nucleolus—one for 5S rRNA and another for 18S, 5.8S, and 25S rRNAs—and the synthesis in the cytoplasm of all r-proteins and trans-acting factors that assist ribosome biogenesis. Upon import of these proteins into the nucleus, the pre-rRNAs undergo complex interconnected pathways of modification, folding, binding to r-proteins, and removal of spacer sequences. Assembly continues upon release of preribosomal particles from the nucleolus to the nucleoplasm and is completed upon export to the cytoplasm (Figure 2).


Figure 2 Cotranscriptional precursor rRNA (pre-rRNA) processing in Saccharomyces cerevisiae. 
(a) As RNA polymerase I transcribes a ribosomal DNA (rDNA) repeat, nascent pre-rRNAs are cleaved cotranscriptionally at the A2 site, releasing a 43S preribosome containing 20S pre-rRNA. The 66S preribosome containing 27SA2 pre-rRNA is released upon completion of transcription. The pre-rRNA processing sites are indicated along the rDNA gene, and the external and internal transcribed spacer sequences are indicated on the nascent transcript. 
(b) The pre-rRNAs then undergo a series of exo- and endonucleolytic cleavages to remove the spacer sequences, finally liberating mature 18S, 5.8S, and 25S rRNAs. Not shown is the flanking 5S gene, transcribed in the opposite direction.

To meet a growing and dividing cell’s high demand for ribosome production, assembly must occur efficiently and with high fidelity. In yeast, more than 200 assembly factors and 77 small nucleolar RNAs (snoRNAs) associate transiently with nascent ribosomes to facilitate these processes. More than 500 assembly factors and 300 snoRNAs have been found in higher eukaryotes . Assembly of properly functioning subunits is enabled by quality-control mechanisms that identify and eliminate improperly constructed particles that might subvert the pool of active subunits . Thus, the ribosome has become a well-studied model to understand assembly and function of the many different RNPs found in cells.

Understanding ribosome biogenesis is important for human health. Because ribosome function is closely tied to the proper growth and proliferation of cells, dysregulation of ribosome biogenesis has profound consequences. Loss-of-function mutations in most r-proteins or assembly factors are lethal in model organisms and presumably embryonic lethal when homozygous in humans.  Structural and functional analyses have revealed that, despite the presence of so many r-proteins, the ribosome is a ribozyme. The immediate environment of the PTC is devoid of r-proteins and functions as an RNA-based catalyst to promote peptide bond formation. What, then, are the roles of the r-proteins? In the 1970s, pioneering work byMizushima&Nomura and Nierhaus & Dohme to reconstitute bacterial ribosomal subunits (r-subunits) in vitro enabled investigations of how r-proteins participate in the assembly of ribosomes. At the same time, bacterial genetic experiments identified mutations in r-proteins that conferred resistance to antibiotics that block specific steps of protein synthesis, suggesting that r-proteins have roles in ribosome function . However, these antibiotics typically bind rRNA, not r-proteins, indicating that r-proteins play more indirect roles in ribosome function, mediated by their cooperative interactions with rRNA. Initial investigations of assembly and function of eukaryotic ribosomes focused mostly on the processing of pre-rRNAs and on the discovery and functional characterization of trans-acting assembly factors, with less emphasis on the analysis of r-proteins. In contrast, the last 10 years have witnessed a significant increase in efforts to systematically investigate the roles of r-proteins in ribosome assembly in yeast and cultured mammalian cells. This progress has been enabled by more powerful tools to analyze how r-proteins work together with assembly factors to drive r-subunit biogenesis, including better methods to inspect pre-rRNA folding and pre-RNP structure. 

WHAT MIGHT THE STRUCTURE OF THE RIBOSOME TELL US ABOUT R-PROTEIN FUNCTION?
High-resolution X-ray and cryo–electron microscopy (cryo-EM) structures of ribosomes from eukaryotes such as yeast, Drosophila, Tetrahymena, plants, and mammals have been invaluable in developing more detailed models for the roles of r-proteins in ribosome structure and function and, with appropriate caveats, ribosome assembly. These structures, as well as those of bacterial and archaeal ribosomes, have revealed that each subunit contains a core of rRNA with globular domains of r-proteins bound at or partially buried below the surface (Figure 1). Perhaps reflecting more diverse regulation of translation in eukaryotes, their ribosomes are larger and more complex than those of prokaryotes, although the common core is highly conserved in all forms of life. There are both eukaryote-specific r-proteins not found in prokaryotes (Table 1) and many more r-proteins in eukaryotes that contain specific tails extending from their globular domains, typically predicted to be intrinsically disordered.











Several of these extensions protrude deep into the rRNA. However, most of them thread across the surface of the subunits and contact multiple domains of rRNA, suggesting that they play a pivotal role in bringing and/or keeping rRNA domains together. In addition to the eukaryote-specific proteins and r-protein extensions, there are extra sequences embedded in eukaryotic rRNA, known as expansion segments (ES). The ES are clustered in several neighborhoods of both the SSU and the LSU, and many contact eukaryote-specific r-protein extensions. Although the structure of each subunit must be established by interactions among the r-proteins and the rRNAs, the importance of such networks of interactions with r-proteins has been tested in only a few cases, which focused more on effects on ribosome function than on assembly. In developing models for the roles of r-proteins in ribosome assembly, the known locations of r-proteins within mature subunits have provided powerful platforms that are guiding our thinking. Although one assumes that the locations of most r-proteins are similar in assembling particles, one keeps in mind examples of the differences discussed in the next sections

LESSONS LEARNED FROM STUDYING IN VITRO RECONSTITUTION OF BACTERIAL RIBOSOMES
Following the initial experiments to reconstitute r-subunits in vitro under equilibrium conditions, investigators studied the kinetics of rRNA folding and r-protein– rRNA interactions with sophisticated biophysical approaches. Although most of these more recent experiments focused on in vitro reconstitution of the SSU, the following principles that emerged guide our thinking about the assembly of both subunits in vivo.
1. In the absence of r-proteins, and in the presence of high Mg2+ concentrations, rRNAs rapidly fold into secondary and tertiary structures resembling those found in mature subunits in vivo. However, there are multiple different rRNA folding pathways, some of which form kinetically trapped or unstable structures.
2. Binding of r-proteins to rRNA helps to overcome these problems by guiding the proper folding of rRNA and by stabilizing productive conformers.
3. In vitro, each rRNA secondary-structure domain of the SSU can fold and bind r-proteins independently of other domains, suggesting that multiple different folding and assembly pathways can be followed.
4. Binding of individual r-proteins to rRNA occurs in stages. The molecules first form labile encounter complexes, followed by generation of one or more intermediates, until the native complex is formed. Thus, initial interactions are weak and then strengthened as assembly proceeds. During these transitions, both the r-proteins and the rRNA can undergo structural changes—a mutually induced-fit mechanism.
5. Association of r-proteins with rRNA has both local and long-range effects on rRNA folding and RNP formation.
6. These conformational changes create hierarchical and cooperative assembly pathways. Association of early, “primary binding” r-proteins organizes binding sites for subsequent assembly of secondary, then tertiary r-proteins. In addition, primary binders tend to associate with the 5' domain of 16S rRNA and tertiary binders with its 3' domain.

Together, these principles reveal that, although largely hierarchical, assembly occurs through multiple parallel and alternative pathways, and that assembly tends to occur in a 5'-to-3' direction with respect to rRNA.

INCORPORATION OF R-PROTEINS INTO NASCENT PRERIBOSOMAL PARTICLES: THE ROLE OF R-PROTEIN IMPORTERS AND CHAPERONES
How are newly synthesized r-proteins imported into the nucleus prior to their association with nascent rRNA? By what means are these abundant, highly basic RNA-binding proteins properly folded into stable, soluble forms and prevented from inappropriately interacting with other cellular RNAs before they assemble into preribosomes? Although most r-proteins are small enough to passively enter the nucleus, due to the high cellular demand of ribosomes, their nuclear import is facilitated by transporters, which recognize their nuclear localization signals (NLSs).  Most nascent NLS-containing yeast r-proteins are imported into the nucleus bound mainly to the β-karyopherin Kap123, a HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and target of rapamycin 1) repeat–containing protein. Kap123 is a nonessential protein; thus, other β-karyopherins (e.g., Kap108, Kap121) have a redundant role in r-protein import. Similarly, r-proteins from mammalian cells also use β-karyopherin as importers. In the nucleus, the cargo r-proteins are released due to the presence of Ran-GTP. How exactly β-karyopherins recognize their cargo r-proteins remains unclear, given that no structural data of any r-protein in a complex with a β-karyopherin are available at atomic resolution. In addition to this general mechanism of nuclear import, specific r-proteins require exclusive systems to be imported to the nucleus. In yeast, the best-characterized system consists of the nonessential symportin Syo1, which associates with the β-karyopherin Kap104; this complex helps simultaneously import r-proteins L5 and L11. The crystal structure of Chaetomium thermophilum Syo1 in complex with the N-terminal extension of L5 has been resolved to atomic resolution. In the nucleus, the Syo1–L5–L11 complex is released from Kap104, as a result of its interaction with Ran-GTP. This action is concomitant with the binding of 5S pre-rRNA. Apparently, association of L5 with Syo1 or 5S rRNA is mutually exclusive, given that the 5S rRNA–binding site of L5 is also localized in the N-terminal extension of L5. In the nucleus, the 5S rRNA–L5–L11 complex (known as 5S RNP) interacts with two assembly factors, Rpf2 and Rrs1, which recruit the 5S RNP to early preribosomal particles. However, it is unclear how Rpf2 and Rrs1 replace Syo1 and how exactly these factors promote stable 5S RNP incorporation into the preribosomal particles. Several other examples of yeast factors that promote efficient recruitment or assembly of specific r-proteins into preribosomal particles have been reported. The WD repeat–containing protein Rrb1 directly and specifically interacts with free L3. It is thought that Rrb1 binds L3 in the cytoplasm and delivers it to nascent pre-60S ribosomes, where L3 stably assembles. In contrast to Rpf2 and Rrs1, Rrb1 only very weakly associates with preribosomal particles. Two other WD repeat–containing proteins, Rrp7 and Sqt1, have been genetically linked to distinct r-proteins. The phenotypic defects of loss-of-function rrp7 mutants are partially suppressed by overexpression of r-protein S27, whereas Sqt1 is a high-copy suppressor of dominant-negative C-terminal truncated mutants of r-protein L10. Sqt1 function is likely similar to that of Rrb1: It stably binds free L10 through the N-terminal part of L10 but weakly binds preribosomal particles. Strikingly, assembly of L10 is likely cytoplasmic; therefore, Sqt1 does not act as a nucleocytoplasmic transporter but rather as an L10- specific chaperone. Consistent with this function, L10 is highly unstable in vivo in the absence of functional Sqt1.

ASSEMBLY OF 40S RIBOSOMAL SUBUNITS
Both pre-rRNA processing and binding of r-proteins to pre-18S rRNA occur largely transcriptionally in the nucleolus (Figure 2a).  Thus, studies of rRNA processing have long served as a proxy for studies of 40S r-subunit assembly. Analyses of rRNA processing in wild-type yeast strains, as well as in many of those depleted individually for trans-acting assembly factors, have revealed that in yeast there are four rRNA cleavage steps on the pathway to producing mature 18S rRNA—two each on the 5' and 3' ends, which first cleave nearby and then at the mature site (Figure 2b). This finding suggests that at least four SSU assembly intermediates exist, one prior to each of the cleavage steps. Nevertheless, in wild-type cells only one pre-40S intermediate accumulates perceptibly; it contains an 18S rRNA precursor referred to as 20S pre-rRNA. This intermediate, formed in the nucleus, contains the mature 5' end, and its 3' end is the A2 site; thus, it contains ∼200 extra nucleotides at the 3' end (Figure 2b). A second intermediate, 23S pre-rRNA, is also found in many yeast strains. This intermediate contains an unprocessed 5' end but has been cleaved at the 3' end, downstream of the typical site, at site A3. Independent sedimentation studies of r-particles have provided evidence for three broad classes of SSU assembly intermediates in wild-type cells. Two early intermediates sediment at ∼90S and contain 23S or 35S pre-rRNAs. In addition, there is a late intermediate, which forms in the nucleus but is located in the cytoplasm at steady state. This late intermediate sediments at 43S and contains 20S pre-rRNA. Finally, the fourth observed intermediate, containing 23S rRNA, sediments at ∼60S. Due to its similar size to the other intermediates and its presumed RNA content, this fourth intermediate may be related to a fifth one observed in cells in which helicase Dhr1/Ecm16 is mutated. Alternatively, the Dhr1-related intermediate may be a dead end observed only in the absence of Dhr1. Because most nascent rRNAs are transcriptionally cleaved at site A2, we do not know whether the presence of 35S pre-rRNA, or even 23S pre-rRNA, is relevant, as these may be degraded before they are mature. Unfortunately, with the exception of the late cytoplasmic 43S assembly intermediate, none of the other assembly intermediates has been purified to sufficient homogeneity to enable either biochemical or structural analyses. Consequently, our knowledge about the complement of assembly factors or r-proteins bound to each consecutive intermediate is tentative. Nevertheless, systematic analyses have shown that r-proteins of the SSU assemble in a bipartite manner and that individual r-proteins fall into three to four broad classes (Table 1) (Figure 3a,b).


Figure 3 Correlation of function and location of the small subunit (SSU) and large subunit (LSU) r-proteins of Saccharomyces cerevisiae. Early-acting ( yellow), middle-acting (blue), and late-acting (red ) r-proteins are mapped onto the crystal structure. (a,c) The subunit interface of the SSU and LSU, respectively. (b,d) The solvent-exposed surface of the SSU and LSU, respectively. Ribosomal RNA (rRNA) and r-proteins are shown in cartoon and surface representation, respectively. Abbreviations: CP, central protuberance; GAC, GTPase-activation center. The crystal structure is adapted from Protein Data bank codes 3U5D and 3U5E.

Generally, r-proteins that bind to the body of the SSU (5' domain) appear to bind early during transcription (Figure 3a,b). Their deficiency blocks assembly and processing at the early cleavage sites (A0 and A1) at the 5' end of 18S rRNA. Because the downstream processing events at A2 and D appear to depend on the prior cleavage at site A1 (and site A2 for D-site cleavage), these are blocked as well. Next, the r-proteins that form the head domain of SSU assemble, and their deficiency impairs mainly cleavage at site A2 (and, as a result, D-site cleavage), which separates the rRNAs destined for the SSU and LSU (Figure 3a,b). Because processing at site A2 also occurs predominantly co-transcriptionally, assembly of this group of r-proteins, which make up 70% of all SSU r-proteins, must also occur co-transcriptionally. Consistent with this idea are the findings that 21 of 22 r-proteins from the SSU that can be analyzed systematically do precipitate significant amounts of 20S pre-rRNA, and only S11 and S13, which bind to the platform, precipitate substantial amounts of 35S rRNA. S26 is the only protein that does not efficiently precipitate 20S pre-rRNA, suggesting that it does not bind until 18S rRNA is mature. Interestingly, the interactions between r-proteins and 35S pre-rRNA are particularly salt labile, suggesting that these r-proteins that bind early pre-rRNAs acquire additional interactions during assembly because of conformational rearrangements of pre-rRNAs, stepwise acquisition of RNA–protein contacts, or the cementing of these contacts by addition of neighboring proteins. A small subset of head-binding r-proteins (S3, S15, S18, and S19) is also specifically required for export of the nascent subunit. Finally, another subset of r-proteins (S10, S20, S26, S29, S31, and Asc1) may assemble in the cytoplasm and is required only for the cytoplasmic processing of 20S pre-rRNA. With the exception of Asc1, these proteins have in common a location near or at the mRNA-binding channel.

MODULATION OF 40S R-PROTEIN BINDING BY ASSEMBLY FACTORS
An emerging theme in ribosome assembly is that there are multiple instances of trans-acting assembly factors that delay the incorporation of r-proteins by sterically blocking their site of assembly. Two such examples are S10 and S26, two of the three last r-proteins to be incorporated into the nascent SSU. Premature binding of S10 to the beak structure is blocked by Ltv1, and binding of S26 to the platform is blocked by Pno1/Dim2. Release of Ltv1 from pre-40S subunits requires its phosphorylation by the kinase Hrr25. How Pno1 is released remains unknown. Interestingly, S10 and S26 are located at the entrance and exit, respectively, of the mRNA-binding channel, suggesting that blocking the binding of these two r-proteins and then regulating the release of the factor (at least in the case of S10) prevent the recruitment of mRNAs to the nascent 40S r-subunit. A comparison between this process and bacterial SSU assembly both in vivo and in vitro also suggests that the platform and beak structure are intrinsically slow to form and initially misfolded. Thus, Ltv1 and Pno1 appear to exploit several already-existing features. First, structural and biochemical analyses of successive in vitro assembly intermediates indicate that the beak and platform regions are the last to assemble r-proteins. Second, an in vitro analysis of rRNA folding has shown that these structures are the slowest to fold. Third, both the beak structure and the platform region are initially misfolded and then refolded during the heat-dependent activation step. Finally, and intriguingly, just as Ltv1 modulates the incorporation of S10 and S3 in eukaryotes, RimM modulates the assembly of the bacterial ortholog of S3 (also known as S3), by regulating folding of 16S rRNA. 

CYTOPLASMIC STEPS OF 40S RIBOSOMAL SUBUNIT MATURATION
The cytoplasmic steps of 40S r-subunit assembly include the incorporation of several r-proteins; formation of the 3' end of mature 18S rRNA; and dissociation of the remaining assembly factors. These events are integrated into a translation-like cycle that serves to “testdrive” nascent ribosomes to ensure that they are competent in key functionalities of the SSU (Figure 4a). 


Figure 4
(a) Cytoplasmic steps of 40S incorporation. (i ) A 40S assembly intermediate containing seven stably bound assembly factors accumulates in the cytoplasm at steady state. Phosphorylation of Ltv1 by the kinase Hrr25 releases Ltv1 and Enp1, allowing for (ii ) repositioning of S3 and incorporation of S10 at the messenger RNA (mRNA) entry channel. (iii ) Release of Ltv1 allows for eIF5B-dependent joining of the 60S subunit to form 80S-like ribosomes. (iv) Before Fap7 acts on 80S-like ribosomes, Rio2 is released, and independently, Asc1 joins. (v) Dim1 is released ( J. Trepreau & K. Karbstein, unpublished data) before (vi ) Nob1-dependent 18S formation and Tsr1 release. (vii ) Tsr1 release allows for binding of Dom34/Rli1 to dissociate 80S-like ribosomes. (viii ) Exchange of Pno1 for S26 occurs in polysomes (97). The nascent 40S subunit is shown in light gray, the mature 60S subunit in dark gray, r-proteins in magenta, stably bound assembly factors in yellow, transiently bound assembly factors/translation factors in green, and mRNA in blue. S3 and S10 are in lighter shades to indicate their location on the solvent side of the molecule. 
(b) Cytoplasmic maturation of pre-60S ribosomal particles. Pre-60S ribosomal particles that arrive in the cytoplasm contain only a few stably bound export adaptors (Arx1–Alb1, Mex67–Mtr2, Nmd3, Bud20) and assembly factors (Mtr4, Nog1, Rlp24, Tif6) that are sequentially released to enable assembly of the remaining r-proteins. Note that the exact order of some steps (e.g., release of Mex67–Mtr2, assembly of L29) has still not been properly addressed. (i ) The first step is the release of Rlp24, Nog1, and Bud20 by the ATPase Drg1, which then permits the assembly of L24 and the recruitment of Rei1. (ii ) Rei1, together with the J protein Jjj1 and the HSP70 ATPase Ssa, enables the release of Arx1–Alb1, located near the polypeptide exit tunnel. Thus, this functional ribosomal site is inactive until the release of Arx1–Alb1. (iii ) Then, or in parallel, Yvh1 is required for the removal of Mrt4, which is replaced in the pre-60S particles by the stalk r-protein P0. The stalk is required for recruitment of translation elongation factors (eEFs); thus, pre-60S particles lacking P0 are inactive. (iv) Pre-60S particles containing P0 are able to recruit the GTPase Efl1, which is closely related to eEF2. Efl1, together with Sdo1, facilitates the release of Tif6 from pre-60S ribosomal subunits (r-subunits). Tif6 inhibits r-subunit joining, thus preventing pre-60S particles from prematurely engaging in translation. (v) The release of Tif6 leads to activation of the GTPase Lsg1 to release the export adaptor Nmd3. Assembly of L40 and L10, aided by the chaperone Sqt1, is also required for the release of Nmd3. Nmd3 binds to the joining surface of the 60S r-subunit, thus also impeding joining with 40S r-subunits. Release of Nmd3 allows the 60S r-subunits to gain translation competence. Finally, acidic r-proteins P1 and P2 assemble to the r stalk at the moment when the mature 60S r-subunit is joined to the 40S r-subunit and committed to translation.

As maturing 40S r-subunits emerge from the nucleus, they contain 20S pre-rRNA and most r-proteins. S20, S29, and S31 are incorporated around the time of export, and at least S20 and S29 appear to be only partially occupied in a purified cytoplasmic intermediate. S10, S26, and Asc1 are completely absent, and S3 and S14 appear not to be positioned at their final location. This assembly intermediate also contains seven stably bound assembly factors: Enp1 and Ltv1, bound to the beak structure; Rio2, Tsr1, and Dim1, located at the subunit interface; and Nob1 and Pno1 on the platform. Phosphorylation of the assembly factor Ltv1 initiates the cytoplasmic maturation cascade by releasing Ltv1 from the beak. Because these two assembly factors hold S3 in a premature conformation and block the binding of S10, their release is expected to lead to final assembly of the mRNA entry channel at the beak structure. Release of Ltv1 is also required for eIF5B-dependent joining of the LSU to initiate the translation-like cycle. The next isolated intermediate is an 80S-like complex that contains pre-40S subunits bound to mature 60S subunits. Rio2 dissociates from this intermediate, and the levels of S10 and Asc1 are akin to those found in mature ribosomes. The exact order of subsequent events remains unclear, but Nob1-dependent 18S rRNA maturation occurs within 80S-like ribosomes and is somehow regulated by the kinase Rio1. Interestingly, although Rio1 cosediments with and copurifies 40S-sized complexes, overexpression of dominant-negative forms leads to the accumulation of 80S-like ribosomes. Furthermore, Dim1 and Tsr1 must dissociate from 80S-like ribosomes. Because Tsr1 and the termination factor Rli1 share a binding site, Tsr1 dissociation allows for the dissociation of 80S-like ribosomes. The newly liberated 40S subunits are now mature, except for the presence of Pno1 and the absence of S26. Note that a recent contrasting study suggested that S26 might be recruited to early nucleolar ribosomes. Clearly, this translation-like cycle has the potential to test the subunit’s ability to bind 60S subunits and to position and activate the translation factors eIF5B and Rli1. Because Rli1 has a cofactor, Dom34, that is also involved in this process and binds the decoding center, this cycle might also test the integrity of this site. Nevertheless, we do not yet know how the successful function of these translation factors is linked to progress in the assembly cascade, including the incorporation of the remaining r-proteins.

ASSEMBLY OF 60S RIBOSOMAL SUBUNITS
Assembly of 60S subunits has been most simply defined by the six successive steps of processing of pre-rRNAs within pre-60S particles (Figure 2b). More recently, assembly factors that are present in these 66S preribosomes during defined intervals of biogenesis have provided additional landmarks for LSU maturation. Whereas assembly of the SSU may occur largely co-transcriptionally, only initial stages of LSU assembly are cotranscriptional. Production of 60S r-subunits is initiated by cleavage of nascent pre-rRNA at the A2 site. This cleavage occurs once transcription by RNA polymerase I has proceeded ∼1–1.5 kb 3' of that site (Figure 2a). Upon completion of transcription, the first detectable precursor particle that is specific for LSU assembly is formed; it contains 27SA2 pre-rRNA as well as most of the r-proteins and approximately one-third of the 75 assembly factors dedicated to making the LSU. Nevertheless, these r-proteins are bound rather weakly and thus have not yet fully formed all of their native interactions with rRNA. As assembly proceeds, association between these r-proteins and preribosomes is strengthened, and downstream steps in pre-rRNA processing occur. In addition, early-acting assembly factors are released, and lateacting assembly factors join nascent ribosomes, then dissociate upon carrying out their respective functions. As described later in this section, an examination of the effects of depleting LSU proteins revealed that assembly of 60S subunits proceeds in a hierarchical fashion, neighborhood by neighborhood. Initially, investigators studied the roles of a few individual r-proteins in LSU biogenesis by depleting them via shutoff of conditional promoter fusions, then assaying effects on cell growth, levels of free r-subunits and polysomes, pre-rRNA processing, and nuclear export of pre-rRNPs. More recently, the roles of most 60S r-proteins have been systematically examined, with assays extended to measuring changes in protein constituents of purified preribosomal particles and, in a few cases, interrogation of effects on pre-rRNP structure. Because most 60S r-proteins associate with the earliest pre-60S particles and contact multiple different intertwined domains of rRNA secondary structure in mature subunits, one might have predicted that depletion of each r-protein would globally affect the earliest stages of assembly. Thus, it was surprising to discover distinct classes of pre-rRNA processing phenotypes upon depletion of 60S r-proteins: Depletion of any of 12 60S r-proteins (L3, L4, L6, L7, L8, L13, L16, L18, L20, L32, L33, and L36) impairs the earliest steps of 27SA2 pre-rRNA processing, as evidenced by the accumulation of 27SA2 or 27SA3 pre-rRNAs and diminished amounts of all downstream pre-rRNAs. Depletion of 11 other LSU r-proteins (L9, L17, L19, L23, L25, L26, L27, L31, L34, L35, and L37) causes accumulation of significant amounts of the next processing intermediate, 27SB pre-rRNA (Table 1). Seven r-proteins (L2, L5, L11, L21, L28, L39, and L43) are required for processing of 7S pre-rRNA or 6S pre-rRNA. just before nuclear export of pre-ribosomes. Depletion of some of the latter proteins also has moderate effects on the processing of 27SB pre-rRNA (Table 1). Finally, depletion of LSU r-proteins that assemble with pre-60S intermediates predominantly in the cytoplasm (e.g., L10, L29, L40, P0, P1, and P2) has little or no direct effect on pre-rRNA processing (Table 1). Strikingly, these classes of pre-RNA processing phenotypes correlate with the location of the corresponding r-proteins within mature 60S r-subunits (Figure 3c,d ). The r-proteins that are necessary for the early steps of 27SA pre-rRNA processing are located on the solvent-exposed surface of LSU, bound primarily to domains I and II of 25S rRNA. The group of r-proteins necessary for the middle steps of pre-rRNA processing clusters in a neighborhood around the exit of the polypeptide exit tunnel (PET), defined by domains I and III of 25S rRNA and 5.8S rRNA. r-Proteins that function in 7S pre-rRNA processing are located on the intersubunit surface, and those required for late nuclear and cytoplasmic steps of maturation cluster around the CP, where 5S rRNAlies between domains II andVofrRNA(Figure 3c,d ). These results suggest that assembly of LSUs may proceed in a hierarchical fashion, beginning with the solvent-exposed surface, followed by the PET, the intersubunit interface, and finally the CP. Importantly, bacterial LSUs assemble with a similar hierarchy, suggesting that the principles governing assembly of LSUs are evolutionarily conserved, despite the added complexities of ribosome biogenesis in eukaryotes. An examination of the effects of these depletions on association of other LSU r-proteins or assembly factors confirmed that there is an assembly hierarchy in which early-acting r-proteins are necessary for stable association of middle-acting r-proteins with pre-rRNPs. Middle-acting r-proteins in turn are required for assembly of late-acting r-proteins. A similar hierarchical dependence upon r-proteins for association of assembly factors with preribosomes was observed. In general, the most affected proteins in these depletion strains are r-proteins and assembly factors located in close proximity to the depleted r-protein. For example, depletion of early-acting L8 bound to domain I of 25S rRNA significantly decreases binding of the adjacent r-proteins L13, L16, and L36, as well as binding of six assembly factors that cross-link to nearby rRNA sequences. Like L8, L13, and L16, these assembly factors are specifically required for processing of 27SA3 pre-rRNA. An analysis of the synthesis and turnover of intermediates that accumulate in the absence of proteins revealed that pre-60S rRNPs become more stable as assembly progresses. Depletion of any one of the early-acting r-proteins leads to rapid turnover of pre-rRNAs. In contrast, when middle-acting r-proteins are depleted, pre-rRNAs turn over less rapidly than in the early class of mutants. Finally, depletion of late-acting r-proteins has a very small effect on pre-rRNA turnover. The increasing stability of pre-rRNPs as they assemble likely reflects the establishment of greater numbers of contacts between the r-proteins and rRNA. Determining how these neighborhoods of the LSU are sequentially assembled, including how pre-rRNA processing is coupled with remodeling of RNP domains within the LSU, remains an important challenge. On the basis of results obtained to date, we present a working model for the pathway of LSU assembly in the following subsections.

Initiating assembly. 
The earliest steps of LSU assembly likely involve bringing together and stabilizing rRNP domains containing both the 5' and 3' ends of 27SA2 pre-rRNA, aided by binding of r-proteins such as L3 to both rRNA domains. In mature subunits, L3 is positioned close to both the 5' end of 5.8S rRNA and the 3' end of 25S rRNA, and it makes the greatest number of contacts with domain VI at the 3' end of 25S rRNA. Thus, L3 may be located in preribosomes near the 5' and 3' ends of 27SA2 pre-rRNA. Furthermore, depletion of L3 has the greatest effect of any LSU r-protein on assembly of pre-60S particles; it causes the earliest block in pre-rRNA processing, leading to a mild accumulation of the 27SA2 pre-rRNA and rapid turnover of nascent particles. A set of assembly factors functionally related to L3 (Npa1/Urb1, Npa2/Urb2, Dbp6, Dpb7, Dbp9, Rsa3, and Nop8) and Rrp5 may participate in this initial compaction of the nascent particles. Each factor, like L3, is important for the processing of 27SA2 prerRNA, and each exhibits genetic interactions with rpl3 mutations. Several other observations support this model. Sequences near the 3' end of yeast pre-rRNA are important for the initiation of processing at site A3 near the 5' end of 27SA2 pre-rRNA. In bacteria, sequences flanking each end of 23S rRNA form a helix that is necessary for production of the mature rRNA. L3 binds to these ends of 23S rRNA and is required to initiate in vitro assembly of the bacterial LSU. Furthermore, in bacteria, domains I and II at the 5' end of 23S rRNA plus domain VI at the 3' end are assembled first, before domains III, IV, and V. Thus, in both prokaryotes and eukaryotes, formation of an initial, compact, pre-LSU intermediate may be an important step for launching assembly of LSUs.

Coupling early steps of large subunit assembly and preribosomal RNA processing with middle steps.
During ribosome assembly, stabilization of initial encounter complexes between r-proteins and rRNA appears to occur in a sequential neighborhood-by-neighborhood fashion, in concert with the binding and function of assembly factors, and may be coupled with pre-rRNA processing. A mutually interdependent association between r-proteins and assembly factors and domains I and II of the rRNA (see Table 1 for these r-proteins) likely allows the formation of an assembly intermediate that is stable enough to carry out the first step of pre-rRNA processing within pre-60S r-particles, namely removal of the remainder of ITS1 from 27SA2 pre-rRNA. Formation of the “bow tie” structure of 5.8S rRNA, by base-pairing of the 5' and 3' ends of domain I rRNA, may be essential for the preparation of a functional substrate for removal of ITS1. Moreover, proper folding of ITS2 and the proximal stem may be a prerequisite for removal of ITS1, indicating tight coupling between the steps for processing of these two spacer sequences. Finally, proper folding of domains I and II may also create a platform for stable formation of downstream rRNA tertiary structures; an inspection of mature LSU rRNA in both prokaryotes and eukaryotes revealed helices in domain II that project toward domains IV and V.


Coupling of middle steps of assembly and preribosomal RNA processing. 
The next step in assembly, strengthening association of middle-acting r-proteins with domains I and III, appears to trigger cleavage of the C2 site in the ITS2 spacer. These r-proteins are necessary to recruit the last two assembly factors required for C2 cleavage, Nsa2 and Nog2/Nug2. Unlike the other middle-acting assembly factors that bind to early-assembly intermediates, Nsa2 and Nog2 assemble immediately prior to C2 cleavage. How they depend on these r-proteins is not clear; their loading may require long-range interactions within pre-60S particles, given that Nog2 binds to helices in domains II, IV, and V on the subunit interface of the pre-60S r-particles, whereas the middle-acting r-proteins bind domains I and III on the opposite side of the subunit. The middle-acting r-proteins are also required for the stable assembly of r-proteins L2, L39, and L43, which are located on the subunit interface adjacent to the PTC. L2 and L43, along with Nog2, are necessary for processing of 7S pre-rRNA. How the stable assembly of these r-proteins is coupled with this step of pre-rRNA processing is unclear, although the effect may be somewhat proximal, given that these r-proteins lie adjacent to the foot structure containing the ITS2 sequences removed in this step, but on different sides of the 60S subunit.

Coupling the late nuclear steps of large subunit assembly with nuclear export.
The last step of LSU biogenesis prior to nuclear export is completion of formation of the CP, which contains 5S rRNA bound to r-proteins L5 and L11. This 5S RNP is delivered into early pre-60S r-particles by assembly factors Rpf2 and Rrs1 (see the section titled Incorporation of r-Proteins into Nascent Preribosomal Particles: The Role of r-Protein Importers and Chaperones, above) but initially is rotated 180◦ from its final position in the mature LSU. Thus, a late step in LSU maturation appears to be rotation of the CP, as well as remodeling of adjacent rRNA helices in the 25S rRNA. L21, which lies at the base of the CP, seems to play at least an indirect role in both CP rotation and nuclear export of pre-60S particles by enabling release of assembly factors Rsa4, Nog2, Rpf2, and Rrs1 (25). L21 may do so by helping structure the adjacent neighborhood containing these factors so that they can be removed by the AAA ATPase Rea1. Release of Rpf2 and Rrs1 bound to the 5S RNP, and of Rsa4, which is present between the CP and the remainder of the pre-60S structure, may be required for rotation of the 5S RNP into its mature position. Reorganization of the CP containing 5S RNP, and release of Nog2, might be coupled to nuclear export of nascent LSUs. Nog2 and the export factor Nmd3 occupy overlapping positions on the
pre-60S particles; consequently, release of Nog2 enables binding of Nmd3. In addition, the export factor Mex67 is thought to bind to the 5S RNP in pre-60S r-particles at this point, perhaps as a result of reorientation of the CP.

CYTOPLASMIC STEPS OF 60S RIBOSOMAL SUBUNIT ASSEMBLY
As for the SSU, the final steps of LSU maturation occur in the cytoplasm, where 5.8S rRNA is generated from 6S pre-rRNA; eight r-proteins, L10, L24, L29, L40, L42, P0, P1, and P2, join the LSU; and seven assembly factors are removed (Figure 4b). These eight r-proteins are specifically enriched in late and cytoplasmic pre-60S r-particles, and none of them are directly necessary for any steps of pre-rRNA processing, even conversion of 6S pre-rRNA to 5.8S rRNA. Furthermore, five of these r-proteins, L24, L29, L41, P1, and P2, are not essential for growth and thus for assembly of translation-competent LSUs (Table 1). All but L24 are located on the subunit interface, adjacent to the CP, the PTC, and the GTPase-activating center (GAC). In the absence of L29 and L40 or upon depletion of L10, the LSU does not efficiently join to the SSUs. Thus, the last steps of LSU assembly are reserved for completion of active sites. Doing so may provide yet another mechanism to prevent inactive, nascent LSUs from interacting prematurely with SSUs. Importantly, the strategy to assemble functional sites of ribosomes last resembles that previously shown for the maturation of bacterial LSUs as well as for yeast SSUs. As is the case for the SSU, premature assembly of r-proteins L10, L24, and P0 into the LSU is sterically blocked by assembly factors bound to late cytoplasmic preribosomes. Removal of these assembly factors is coupled with binding of the corresponding r-proteins, as part of an ordered pathway of cytoplasmic maturation of the LSU (Figure 4b). This pathway is carried out by a series of GTPases and ATPases that are recruited to and activated by factors present in late cytoplasmic pre-60S particles, triggering release of their respective target proteins and, in some cases, association of the corresponding r-protein. Each step in this pathway appears to be coupled to the next downstream remodeling event. The first factor that functions in cytoplasmic maturation of pre-60S particles is the AAA ATPase Drg1, which is recruited and activated by the assembly factor Rlp24, a paralog of protein L24. Drg1 catalyzes the removal of Rlp24, after which L24 joins the cytoplasmic pre-60S subunits. This step enables binding of Rei1, which together with the HSP70 ATPase Ssa1 and its cofactor Jjj1 releases the export adaptor Arx1 and its partner Alb1. In parallel, r-protein L12 recruits the assembly factor Yvh1, which displaces Mrt4, the paralog of r-protein P0, from its rRNA-binding site in the GAC. This reaction enables the irreversible binding of P0. In contrast, the P1 and P2 r-proteins, which together with P0 form the stalk structure, cycle on and off of mature 60S subunits. Assembly of the P stalk is necessary for binding and activation of the GTPase Efl1. It is during these last steps of subunit maturation that the LSU undergoes functional proofreading to test the assembly of the tRNA P site adjacent to the PTC and the GAC. It is thought that the flexible loop of L10, which is positioned adjacent to the P site in translating ribosomes, detects whether or not assembly of the PTC occurs properly and, if so, transmits signals to activate Efl1 and Sdo1, thereby releasing Tif6. This process then triggers release of Nmd3 by Lsg1. In addition, Efl1 binds to and is activated by the P stalk. Thus, Efl1 might couple proofreading of the functionality of the LSU with removal of factors that prevent nascent LSUs from interacting with 40S subunits. Thereafter, newly made LSUs can enter the pool of functioning subunits. Intriguingly, Efl1 is structurally related to translation factor eEF2, suggesting that it carries out a step resembling the translocation function of eEF2. It is also tempting to speculate that the translation-like cycle that functions in 40S subunit assembly might similarly be used to proofread nascent 60S subunits. eIF5B, the key functional player, might be as unable to distinguish between mature 60S and pre-60S ribosomes as it is unable to distinguish between 40S and pre-40S ribosomes.

Assembly of the bacterial ribosome

To guarantee the efficient assembly of ribosomes, cells are faced with the enormous logistic challenge of producing equal amounts of the four rRNAs and the 79 r-proteins  The transcription of rDNA and r-protein-encoding genes (RPGs) is connected to growth conditions, which are relayed via the TORC1 kinase, but a balanced production is complicated because the transcription of these components includes all three RNA polymerases. A negative feedback loop links early assembly events with  ribosomal protein gene (RPG) transcription.13

My comment: This shows how Ribosome biogenesis is an interdependent process, where transcription, the TORC1 pathway 14, feedback loops, post-transcriptional events, and all the assembly proteins and cofactors work together in a dynamic process.

Both the quantitative and qualitative production of r-proteins clearly depends also on several post-transcriptional events, including the stability and splicing efficiency of ribosomal protein genes (RPG), mRNAs as well as the folding and intrinsic stability of r-proteins and, for most of these, their transport into the nucleus. Owing to these properties, r-proteins are especially prone to aggregation and general ribosome-associated chaperone systems contribute to their soluble expression. A heterogeneous class of proteins, collectively referred to as dedicated chaperones, specifically protects individual r-proteins and safely guides them to their assembly site on preribosomal particles.

My comment: If chaperones where not preventing from the nascent newly synthesised polypeptide chains from aggregating into nonfunctional structures right from the beginning, a functional ribosome would never be assemble and becoming functional. That means, both, the chaperones had to be there to help preventing nondesired aggregation right from the beginning.
 
Notably, the majority of these already capture their r-protein clients during translation.

My comment: This is another noteworthy observation. The prevention has to occur right after the new polypeptide chains are synthesized, otherwise aggregation can occur right away.

The ubiquitin–proteasome system rapidly degrades unincorporated r-proteins, thereby antagonizing their aggregation bias. Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases.

My comment: And this is another system that had to be present right from the beginning, otherwise there would not have been a mechanism to prevent the accumulation of nonfunctional polypeptides, which would jumble the cytosolic space and eventually kill the cell.

To avoid targeting pre-ribosomes for degradation, this nuclear quality control system, termed excess r-protein quality control (ERISQ), specifically ubiquitinates those lysine residues of r-proteins that are no longer accessible after their assembly into preribosomes. Kinetic competition for r-proteins between ERISQ and the preribosome might constitute a mechanism to selectively eliminate excessive r-proteins. Furthermore, association with dedicated chaperones and/or importins may prevent r-proteins from being ubiquitinated, thereby enabling their storage until they are incorporated into preribosomes.

r-Proteins are important not only for the structure and function of the ribosome but also for its assembly. Whereas severe mutations in r-proteins will produce non-functional ribosomes, milder mutations may cause biogenesis and/or translation defects. In humans, reduced translation efficiency or accuracy may lead to ribosomopathies, whose clinical characteristics often include anemia or developmental defects. Moreover, defects in ribosome assembly increase the levels of free r-proteins, acting as a signal for ribosome biogenesis stress. Accordingly, in multicellular organisms, elevated levels of uL5 and uL18 are functionally linked to the E3 ubiquitin ligase MDM2 leading to accumulation of its substrate p53, thereby blocking cell division. Taking these findings together, the levels of free r-proteins are well regulated and monitored by various mechanisms, such as transcriptional regulation, association with binding partners, and degradation, to embed ribosome assembly into other cellular pathways that exert growth control.




RIBOSOME BIOGENESIS IN CELL GROWTH, DISEASE AND AGEING
Ribosome biogenesis is an essential major metabolic process in all organisms. The making of ribosomes in eukaryotes requires the coordinated action of all three RNA polymerases, numerous small nucleolar RNAs (snoRNAs) and several hundred protein factors. The highly dynamic and complex pathway of ribosome synthesis is directly or indirectly linked to various celullar processes. 12



As a high energy demanding process ribosome biogenesis is tightly controlled with respect to the cell growth. A dysregulation of ribosome biogenesis leads to surprisingly tissue-specific diseases and developmental defects. An upregulation of ribosome synthesis is a hallmark of most cancers and consequently the nucleolus and ribosome biogenesis are currently recognized as important targets for cancer therapy. Intriguingly, dysregulation of ribosome synthesis or translation also affects longevity and ageing.

The Tor signaling pathway controls ribosome biogenesis at different levels. While the control of transcription by Tor pathways is mechanistically fairly well understood, the regulation of pre-rRNA processing and assembly at the post-transcriptional level remains largely unexplored.  Yeast switches rapidly between two alternative pre-rRNA processing pathways in response to stress or nutrients availability. 

Ribosome biogenesis is a complex, dynamic process involving the coordinated transcription, processing, modification, and structural remodeling of immature ribosomal RNA (rRNA) and binding of ribosomal proteins.  In eukaryotes, ribosome assembly spans three cellular compartments, beginning in the nucleolus and continuing in the nucleoplasm, with final stages of maturation occurring in the cytoplasm. To ensure efficient and accurate construction of ribosomes, eukaryotic ribosome assembly is facilitated by several hundred protein assembly factors (AFs), which include nucleases, RNA helicases, nucleoside triphosphatases, and scaffolding proteins, among others. 11 Ribosome assembly is hierarchical, with primary binding r-proteins participating in the formation of binding sites for later-entering r-proteins. Large ribosomal subunit assembly occurs in blockwise parallel pathways. As assembling pre-60S subunits progress from the nucleolus to the nucleoplasm, major compositional changes occur as many early-acting assembly factors AFs dissociate from the preribosome and additional AFs bind. During this period of assembly, removal of the internal transcribed spacer 2 (ITS2) RNA is initiated by cleavage of 27SB pre-rRNA at the C2 site by the endonuclease Las1. Las1 functions in a complex with Grc3, Rat1, and Rai1, and both Las1 and Grc3 are required for cleavage at the C2 site. C2 cleavage is coordinated with transit of the pre-60S subunit from the nucleolus to the nucleoplasm by an unknown mechanism.



The sheer number of different molecules the cell must produce to make the subunit is remarkable: The core is a long strand of RNA, and 20 different proteins must be attached to the strand. These get organized by the weak chemical forces between the protein molecules and the RNA -- repelling at some points and attracting in others -- and the whole structure thus relies on the proper manufacture and organization of each component. Add to that another six proteins that are not part of the structure, but act as chaperones to assist in the assembly. That makes at total of a least 27 different genes -- one to encode each component or chaperone -- that must work together to make the subunit.  The subunit assembly is a hierarchal process. 10

Ribosome biogenesis is the process that leads to the assembly of translationally competent ribosomes. It is considered the most energy demanding mechanism in cell metabolism, accounting for more than half of a cell’s whole energy. Ribosome biogenesis starts in a dedicated organelle: the nucleolus, where ribosomal components accumulate. From the transcription of ribosomal RNA to the assembly of the last ribosomal proteins on the 80S ribosome, a plethora of maturation and assembly steps is required. These occur sequentially during the entire journey from the nucleolus fibrillar centre to the cytoplasm.

Biogenesis of the mature bacterial ribosome requires the stepwise association of ∼50 ribosomal proteins and intricately folded rRNA, and involves ∼100 molecular chaperones. Each bacterium can generate ∼100,000 ribosomes per hour 8

The translation machinery is, by far, the most complex part of a modern minimal cell, both in its biogenesis and its function. Therefore, it was not surprising that half of the previously classified as poorly characterized genes have been associated with the maturation of the translation apparatus.

Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. 15  The biosynthesis of ribosomes is, therefore, an essential process for all living organisms. A highly complex interaction of a multiplicity of non-ribosomal proteins and small nucleolar RNAs (snoRNAs) facilitates ribosome formation. Prokaryotic ribosome synthesis is a complex, multistep process requiring the coordinated synthesis, cleavage, post-transcriptional modification and folding of ribosomal RNA (rRNA), and the translation, post-translational modification, folding and binding of approximately 50 ribosomal proteins (r-proteins). 16  Ribosome biogenesis is energetically costly, with the majority of cellular transcription and translational capacity dedicated to the production of new ribosomes.   This process is both rapid, requiring ∼2 minutes for production of a single ribosome, and efficient, with the vast majority of assembly events resulting in mature, translationally active complexes. The assembly of ribosomes is tightly regulated in a growth-rate–dependent manner primarily at the level of rRNA synthesis 7


In the last step of the gene expression pathway, genomic information encoded in messenger RNAs is translated into protein by a ribonucleoprotein called the ribosome.
The bacterial ribosome is an extremely complex macromolecular machine capable of performing a fundamental and instrumental cellular process, protein translation. The ribosome comprises two ribonucleoprotein subunits.


This is a 3D computer graphic model of a ribosome.
Ribosomes are composed of protein and RNA. They consist of subunits that fit together and work as one to translate mRNA (messenger RNA) into a polypeptide chain during protein synthesis (translation).

The ribosome is an essential ribonucleoprotein enzyme, and its biogenesis is a fundamental process in all living cells. 1  

Ongoing investigations are focused on elucidating the cellular processes that facilitate biogenesis of the ribosomal subunits, and many extraribosomal factors, including modification enzymes, remodelling enzymes and GTPases, are being uncovered.

The large, or 50S, subunit, which is responsible for the catalysis of peptide bonds linking the amino acid building blocks of protein, contains two RNA molecules, 23S and 5S, as well as 34 ribosomal proteins (r-proteins).

The small, or 30S, subunit (SSU) contains a 16S RNA molecule and 21 r-proteins and is the site of mRNA decoding.



The complete atomic structure of the large ribosomal subunit at 2.4 A resolution
The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. 3
It is composed of 3045 nucleotides and 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure.



The tertiary and secondary structures of the RNA in the H. marismortui large ribosomal subunit and its domains. 


Large-subunit proteins from Haloarcula marismortui

Structure of the 30S ribosomal subunit
Protein synthesis is a complex, multistep process that requires, in addition to the ribosome, several extrinsic GTP-hydrolysing protein factors during each of the main stages of initiation, elongation and termination. The 30S ribosomal subunit has a crucial role in decoding mRNA by monitoring base pairing between the codon on mRNA and the anticodon on transfer RNA; the 50S subunit catalyses peptide bond formation. 4



Overview of the 30S structure



Structure and dynamics of bacterial ribosome biogenesis
The assembly and maturation of the ribosomal subunits are very complex and involve a series of events, such as processing and modification of rRNA, ordered binding of ribosomal proteins and metal ions, and sequential conformational changes. In vivo, this takes approximately 2 min at 37°C 5

The bacterial ribosome consists of three rRNA molecules and approximately 55 proteins, components that are put together in an intricate and tightly regulated way. Formation of the ribosomal particle involves a complex series of processes, i.e., synthesis, processing and modification of both rRNA and ribosomal proteins, and assembly of the components. The quality of the particle must also be checked and the amount of active ribosomes monitored. All of these events must be tightly regulated and coordinated to avoid energy losses and imbalances in cell physiology. 5

The small subunit, 30S, is made of 16S rRNA (1,542 nucleotides) and 21 ribosomal proteins (r-proteins), while 
the large subunit,  50S, is composed of two rRNAs, 23S (2,904 nt) and 5S (120 nt) rRNA, and 33 proteins. 

Nucleolytic processing of rRNA


Schematic drawing of the rrnB operon.
(A) Nucleolytic processing of the rrnB primary transcript. The rRNA and tRNA species, promoters P1 and P2, and terminators T1 and T2 are indicated, as well as the processing sites of RNase III (III), RNase G (G), RNase E (E), RNase P (P), RNase T (T), and the unknown RNases (?).
(B) Promoter region of the rrnB operon. Locations of FIS- and H-NS-binding sites and the UP, discriminator, and nut sequences are marked. Arrows show the start sites of transcription.

The biogenesis of ribosomes begins with transcription of the 16S, 23S, and 5S rRNA, which are synthesized as one primary transcript (Figure above A). Maturation of the transcript begins before transcription is completed, with instant formation of local secondary structures and, as soon as their binding sites emerge from the polymerase, binding of ribosomal proteins. Simultaneously, rRNA becomes chemically modified at a number of positions and is processed by several RNases to generate mature rRNA species.

After the ribosome structures: How are the subunits assembled?
As if the intricacy of the RNA fold in the ribosome was not overwhelming enough, we are left to grapple with the question “By Jove, how does this thing get put together?” There is a vast amount of information concerning bacterial ribosome assembly. 6

Ribosome biogenesis in Escherichia coli begins with transcription of the ribosomal RNA operon, where the three ribosomal RNAs are synthesized as a single transcript. The subsequent steps surely begin before the entire transcript is completed. It is likely that extensive local secondary structure in the rRNA forms very quickly, and that ribosomal protein binding begins as the protein binding sites are completed. The rRNA transcript is chemically modified at a number of points, and it is processed by nucleolytic cleavage to ultimately generate the 16S, 23S, and 5S chains. 

The assembly process is a carefully choreographed series of RNA conformational changes, protein binding, ion binding, and processing events that occurs cotranscriptionally.

Bacterial ribosome biogenesis has been an active area of research for more than 30 years. Prokaryotic ribosome synthesis is a complex, multistep process requiring the coordinated synthesis, cleavage, post-transcriptional modification and folding of ribosomal RNA (rRNA), and the translation, post-translational modification, folding and binding of approximately 50 ribosomal proteins (r-proteins). Ribosome biogenesis is energetically costly, with the majority of cellular transcription and translational capacity dedicated to the production of new ribosomes. This process is both rapid, requiring 2 minutes for production of a single ribosome, and efficient, with the vast majority of assembly events resulting in mature, translationally active complexes. The assembly of ribosomes is tightly regulated in a growth-rate–dependent manner primarily at the level of rRNA synthesis, which, in turn, regulates r-protein synthesis through an elegant autoregulatory feedback translational control mechanism that prevents the r-proteins’ levels from exceeding the availability of rRNA. The net effect of this network of regulatory mechanisms is a linear relationship between cellular growth rate and ribosomal content that optimally allocates the proteome between catabolic enzymes that produce amino acids and ribosomes that convert these precursors into new biomass.

A co-transcriptional assembly process 
Despite the central cellular role of ribosome assembly in cell physiology and decades of study, the details of ribosome biogenesis are only beginning to emerge. There has been a trade-off between the detailed studies that can be accomplished by studying assembly in vitro, and less precise but more biologically relevant studies that can be carried out in cells. The primary difference between the in vitro and in vivo studies is the co-transcriptional assembly of rRNA that occurs in cells. Direct evidence for co-transcriptional assembly arose from observation of ultrastructure in the rRNA operons, known as ‘Miller spreads’. Miller found that gently fixing the chromatin of rapidly growing cells cross-linked the RNA polymerase (RNAP) and nascent rRNA transcripts, and that these preparations could be visualized by negative stain electron microscopy (EM), as shown in figure below. 


Co-transcriptional assembly of ribosomes in the E. coli ribosomal RNA operon. 
The negative-stain electron micrograph of fixed chromatin shows the high transcriptional level of the ribosomal operon, where over a hundred RNAP molecules can be seen over the 5.5 kb segment. The organization of the rRNA genes is shown below, where the primary rRNA precursor transcript is processed by a series of endonucleolytic cleavage reactions to produce the mature 16S, 23S and 5S rRNAs. The series of increasingly long nascent transcripts can be seen progressing from left to right, with evidence for co-transcriptional binding of ribosomal proteins. In addition, co-transcriptional rRNA processing is observed, indicated by the arrow, which liberates a pre-30S ribonucleoprotein complex prior to initiating the transcription of the 23S rRNA gene.

These images revealed high packing density of RNAP on the rRNA gene with clear evidence for association of additional protein components to the nascent rRNA as the polymerase moved through the operon. Furthermore, a discrete transition from long to short nascent chains at the approximate position of the 30 -terminus of the 16S rRNA coding region provided strong evidence for co-transcriptional rRNA cleavage and release of a 30S precursor early during transcription of the pre-23S rRNA (figure above ).

Assembly guided by RNA folding 
The assembly of the small ribosomal subunit (SSU) requires RNA folding and compaction, which occurs through both r-protein-dependent and independent events.   Ribosome assembly is effectively an RNA folding problem, wherein proteins are used to ‘lock in’ productive RNA folding and drive the structure towards its mature conformation. Much of the native rRNA secondary structure is formed in a protein-independent manner, whereas native tertiary rRNA contacts are often stabilized through protein-binding events.

In vitro ribosomal protein assembly maps 
Despite the highly complex and co-transcriptional nature of the assembly process, active subunits can be reconstituted in vitro using purified r-proteins and rRNA. While these in vitro assembly reactions are generally less efficient than the process in vivo, the success of in vitro assembly implies that the folding determinants are primarily encoded in the ribosomal components themselves. Extensive in vitro reconstitution experiments directly tested the interdependence of r-protein binding. By withholding specific r-proteins, they could classify the r-proteins into groups that bound to rRNA independently (primary binders) and those whose binding was improved by the addition of other r-proteins (secondary binders). These distinct protein-binding classes were the first evidence that ribosome assembly consists both of sequential and parallel processing steps.

The role of ribosome biogenesis factors 
Dozens of accessory biogenesis factors help guide the assembly process, including GTPases, rRNA modification enzymes, helicases and other maturation factors.

These are basically molecular machines required and helping the assembly of the Ribosome factory. These enzymes, evidently, would have no function unless the higher goal, that is to make functional ribosomes, is foreseen as a distant goal. 

Interestingly, many ribosome assembly factors indicate that redundant factors are present or that they are only necessary under particular environmental conditions like cold-stress.

Structure and composition of assembly intermediates 



Cooperative folding blocks of the bacterial large (LSU) and small (SSU) ribosomal subunits. 
(a) Heatmap of median folding block occupancy in various published SSU assembly intermediate structures. Blocks 1 – 4 were derived from hierarchical clustering of the calculated occupancy for each r-protein or rRNA helix across the set of available SSU assembly intermediate structures. 
(b) 16S secondary structure coloured and labelled by domain. Folding blocks as defined by hierarchical clustering in the electronic supplementary material, figure S1, are outlined and coloured according to (a). 
(c) SSU structure model (PDB: 4ybb) with blocks labelled and coloured according to (a). 
(d) Heatmap of median folding block occupancy in various published LSU assembly intermediate structures. Blocks 1 – 6 were derived from hierarchical clustering as in (a) using the set of available LSU assembly intermediate structures (electronic supplementary material, figure S2). 
(e) 23S secondary structure coloured and labelled by domain and folding blocks as in (b). (f ) LSU structure model (PDB: 4ybb) with blocks labelled and coloured according to (d).



Bringing the maturation of ribosomal subunits into focus.
Illustrated are the biogenesis pathways of the functional bacterial ribosomal subunits 30S; RNA colored gray and r-proteins colored blue) and 50S  RNA colored gray and r-proteins colored purple). The biogenesis of these subunits commences with transcription of primary rRNA transcripts, which contain 16S, 23S and 5S rRNA sequences and intervening sequences, and proceeds through a series of ill-defined stages. Although a precise X-ray crystal structure of the ribosomal subunits has been solved, the cellular processes involved in ribosome maturation (presented within the gray box) remain incompletely understood. It is known that an array of ribosomal biogenesis factors facilitates processes through a coordinated series of maturation events. Some of these factors are listed in the middle of the gray box, but the temporal aspects of their function have not been resolved. Elucidating a more complete understanding of the specific processes involved in the biogenesis of ribosomes remains a formidable challenge in the field but would result in a lessening of the ‘gray haze’ overlaying the biogenesis cascade at this time.



. (A) Pre-rRNA processing scheme in Saccharomyces cerevisiae. (B) Pre-rRNA processing scheme in HeLa cells.


Pre-ribosomal particles along the 40S assembly pathway. 
The major intermediates of 40S pre-ribosomes, their rRNA content (dark green) and the presence of ATP/GTPconsuming enzymes are depicted (DExD/H-box ATPases in green, kinases in light blue, GTPase in orange). The nascent 35S pre-rRNA is modified and folded to form a first precursor of the 40S subunit. The cleavages at site A0, A1, A2, which generate the 20S pre-rRNA, are accompanied by a major exchange of non-ribosomal factors. The formation of the beak and final 20S processing occurs in the cytoplasm. 7


Pre-ribosomal particles along the 60S assembly pathway. 
The different 60S pre-ribosomes are depicted together with their rRNA content (blue). The presence of ATP/GTPconsuming enzymes (GTPases in orange, DExD/H-box ATPases in green, AAA-type ATPases in pink), prominent subcomplexes (purple/yellow), and export factors (red) is shown. Bait proteins purifying the corresponding, distinct particles are indicated on top.



https://www.genome.jp/dbget-bin/www_bget?ko03008

“a multitude of transient assembly factors” regulate and systematically fold the proteins that will be used to construct the machine. The authors mention “21 ribosome assembly factors that stabilize and remodel” the RNA and proteins before the machine is even operational. Inside the growing ribosome, a scaffold holds factors for the exit tunnel in place. Everything is choreographed in time and space with “mechanisms driving… assembly in a unidirectional manner.”

Here we see numerous parts working together on a timeline. The parts alone do not work individually. You can have all the proteins delivered to the construction site, and nothing will happen without the programmed mechanisms to put them together in order. Some parts hold others in place, others guide the folding of protein parts, and some even prevent premature assembly. All the pathways for assembly of the subdomains are regulated by a master program, so that each group of steps follows a “unidirectional” plan toward the finished product.
https://evolutionnews.org/2018/03/irreducible-complexity-in-molecular-machine-assembly/

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes
Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage. These proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps. 11

The ribosome is a vital machine in every cell. It is stunning in the details required to make this machine. I like the insight gained through new research. When it is made "It doesn't just go plop". It is a multistep process requiring perfect precision and timing.
Not something unintelligent processes can produce!!
Again glory to God for life!!

"The synthesis of ribosomes is therefore an extremely complex, multistep process, which includes both assembly and maturation stages."

"The "emergence" of the 40S subunit entails an ordered series of reactions in which the outer shell of the 90S particle is progressively dissociated from the 40S. "It doesn't just go plop," Beckmann remarks. The process is actually reminiscent of the molting of an insect—shedding of the integument takes place layer by layer. "It's rather like those Russian dolls. When you open one, you find a smaller one nestled inside," says Beckmann"

https://phys.org/news/2020-09-ribosomes-russian-dolls.html?fbclid=IwAR0YcGlXqObFJAO3_RGxFNUIx271iXe43ljT4gPx-AhBqq2UxIWjbPWbvxc

Ribosome Biogenesis in Eukaryotes
Eukaryotic ribosomes are the 80S ribosomes, consisting of 60S large subunit and 40S small subunit. The large subunit consists of three types of rRNAs (25S in plants or 28S in mammals, 5.8S, and 5S) and about 47 ribosomal proteins. The small subunit consists of one rRNA (18S) and about 33 ribosomal proteins.

Ribosome Biogenesis in Prokaryotes
70S ribosomes, which consist of 50S large and 30S small subunits, are prokaryotic ribosomes. The large subunit consists of two types of rRNAs (23S and 5S) and 33 ribosomal proteins. The small subunit consists of only 16S rRNA and 20 ribosomal proteins.

52 genes of the prokaryotic genome encode the ribosomal proteins. They belong to 20 different operons. Transcription of the ribosome gene operons occurs in the cytoplasm. The synthesis of ribosomal proteins as well as the assembly of large and small subunits of the ribosome occurs in the cytoplasm in prokaryotes.

Epigenetic control of ribosome biogenesis homeostasis 2

1. https://pediaa.com/what-cellular-structure-is-responsible-for-manufacturing-ribosomes/
2. https://tel.archives-ouvertes.fr/tel-01878354/document


Ribosome biogenesis: the essentials. 
The mature 80S ribosome is composed of a 40S subunit containing 18S ribosomal RNA (rRNA) and 33 ribosomal proteins (RPs) and a 60S subunit containing 5S, 5.8S and 28S rRNAs and 47RPs. The majority of steps in ribosome biogenesis occur in the nucleolus, where RNA polymerase I (Pol I) transcribes the 47S precursor rRNAs (47S pre-rRNAs) from ribosomal DNA (rDNA) genes, which contain the sequences of 18S, 5.8S and 28S rRNAs. The 47S prerRNA is co-transcriptionally assembled into the 90S processome with 5S rRNA, which is transcribed by RNA polymerase III (Pol III) in the nucleoplasm, and RPs, for which RP mRNAs are transcribed by RNA polymerase II (Pol II) in the nucleus and exported to the cytoplasm for translation into RPs, which are subsequently re-imported in the nucleolus. During the maturation of the 90S processome into pre-40S and pre-60S ribosomal subunits, pre-rRNA is modified and processed through mechanisms that involve ~200 small nucleolar RNAs (snoRNAs), which are mainly transcribed from introns of other Pol II-driven genes in the nucleoplasm. A few additional RPs are assembled into pre‑40S and pre‑60S ribosomal subunits in the nucleoplasm and the cytoplasm. The complete process of ribosome biogenesis involves several hundred accessory factors, giving rise to the mature 80S ribosome. In the figure, rRNA modifications are indicated by orange pentagons. RPL, large subunit ribosomal proteins; RPS, small subunit ribosomal proteins; 5ʹTOP, 5ʹ-terminal oligopyrimidine tract. 6


Schematic diagram of ribosome biogenesis and MYC-dependent regulation of rRNA synthesis.
The diagram gives a synopsis of the steps involved in ribosome biogenesis and CAP-dependent translation with emphasis to the limiting step, rRNA synthesis.


https://jcs.biologists.org/content/joces/126/21/4815.full.pdf



1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711711/
2. https://royalsocietypublishing.org/doi/pdf/10.1098/rstb.2016.0181
3. https://sci-hub.tw/https://www.ncbi.nlm.nih.gov/pubmed/10937989/
4. https://sci-hub.tw/https://www.ncbi.nlm.nih.gov/pubmed/11014182/
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168646/
6. https://sci-hub.tw/https://www.ncbi.nlm.nih.gov/pubmed/12554857/
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311925/
8. https://www.nature.com/articles/nrmicro.2016.200
9. https://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S0167488909002651?via%3Dihub
10. https://www.sciencedaily.com/releases/2020/05/200521102052.htm
11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028539/
12. https://www.bzh.db-engine.de/group/56/martin%20koš/setLang=en
13. https://core.ac.uk/download/pdf/208158691.pdf
14. https://en.wikipedia.org/wiki/MTORC1[/color]

A bacterial checkpoint protein for ribosome assembly moonlights as an essential metabolite-proofreading enzyme 04 April 2019
https://www.nature.com/articles/s41467-019-09508-z
The ribosome is an abundant and exceptionally complex structure whose assembly and function dominates bacterial physiology. During rapid growth of bacteria such as Bacillus subtilis, the majority of RNA polymerase is engaged in the synthesis of ribosomal RNA (rRNA). Ribosomes comprise up to 50% of cell mass, and translation consumes up to 2/3 of cellular energy. As expected for such an energetically demanding process, ribosome synthesis and assembly is highly regulated, and defects can impose severe fitness costs on cells.

Ribosome assembly requires the efficient processing of the precursor rRNA transcripts followed by the ordered assembly of ribosomal proteins to generate the 30S and 50S subunits. Assembly of the ribosome occurs rapidly in vivo, in a process facilitated by RNA helicases, rRNA modification enzymes, and ribosome-assembly GTPases (RA-GTPases). The RA-GTPases are universally conserved proteins that couple GTP hydrolysis to specific checkpoints in assembly. In B. subtilis, the six RA-GTPases include RbgA, YphC, and YsxC implicated in assembly of the 50S subunit and Era, YqeH, and CpgA to facilitate assembly of the 30S subunit. Of these six RA-GTPases, four are essential and mutants in yqeH and cpgA are growth impaired. CpgA (circularly-permuted GTPase) is also important for normal cell morphology, proper deposition of the peptidoglycan sacculus, and intrinsic resistance to antibiotics affecting both the ribosome and cell wall synthesis. Whether or not these various phenotypes are related to the ribosome-assembly role is not resolved.

CpgA is also a target for PrkC, a eukaryotic-like Hanks Ser/Thr kinase with surface-exposed penicillin binding protein and Ser/Thr kinase associated (PASTA) domains implicated in muropeptide sensing. Indeed, cpgA is co-transcribed with prkC and prpC, encoding the cognate phosphatase for PrkC14. Using phosphomimetic and phosphoablative variants of CpgA, it has been proposed that phosphorylation of CpgA at Thr166 increases intrinsic GTPase activity, enhances association with 30S ribosomal subunits to aid in ribosome maturation, and is necessary for normal cell morphology. However, whether CpgA phosphorylation affects its activities related to peptidoglycan deposition and antibiotic sensitivity is not clear.

Ribosome Biogenesis: An Overview
Ribosome biogenesis is a highly dynamic process in which transcription of the runes, processing/modification of the runes, association of ribosomal proteins (RPs) to the pre-runes, proper folding of the pre-runes, and transport of the maturing ribosomal subunits to the cytoplasm are all combined. In addition to the ribosomal proteins RPs that represent the structural component of the ribosome, over 200 other non-ribosomal proteins and 75 snoRNAs are required for ribosome biogenesis. The final product is a functional ribosome, which, in eukaryotes, consists of 40S and 60S subunits that contain 4 species of processed ribosomal RNAs (18S, 28S, 5.8S, and 5S) and 79 ribosomal proteins (RPs). The mature 40S subunit consists of 18S rRNA and 33 RPs, while the 60S subunit consists of 28S, 5.8S, and 5S runes and 43 RPs. 3



https://pt.3lib.net/book/2850246/630655

Ribosome biogenesis initiates around nucleolar organizing regions, which contain several hundred copies of the ribosomal DNA (rDNA) genes. In humans, these genes are arranged as head-to-tail palindromes on chromosomes 13, 14, 15, 21, and 22 and encode the 47S pre-rRNA transcripts that will later be processed into 28S, 18S, and 5.8S runes. In addition, on a portion of chromosome 1 (human), in association with the nucleoplasm, there are several hundred copies of the 5S rDNA gene. The process begins with the association of the upstream binding factor (UBF) and selectivity factor (SL)-1 to the 47S rDNA promoter. This recruits the RNA polymerase I-specific initiation factor RNN3 (TIF-IA) and RNA pol I to the promoter. The RNA pol I complex formation is assisted by the association of the MYC:MAX heterodimer to upstream E-box elements and the binding of additional regulatory factors, which recruit the histone acetyltransferase (HAT) complex, and can be inhibited by the association of p53 or the pRB/p130 complex to key proteins of the RNA pol I initiation complex. At the same time, TF-IIIA, TF-IIIB, TF-IIIC, and RNA pol III associate with the 5S rDNA promoter. The association of TF-IIIA represents the first step in the assembly of the pol III complex, by both inducing a minor bend in the DNA as well as assisting in the incorporation of TF-IIIC into the polymerase complex. TF-IIIB, in turn, induces a major bend in the DNA at the transcriptional start site. Again, this complex formation is assisted by the association of MYC with TF-IIIB, in the absence of MAX, and the recruitment of the HAT complex, and either p53 or the pRB/p130 complex can suppress RNA pol III-mediated transcription. The 18S, 5.8S, and 28S runes are transcribed by RNA pol I as a single precursor RNA from tandem repeats of the gene into the nucleolus, while the 5S rRNA, which is transcribed by RNA pol III from multiple genes into the nucleoplasm, migrates to the nucleolus. RNA pol III is also responsible for the transcription of tRNA genes needed later for translation initiation and elongation. In contrast, the ribosomal proteins, which are present throughout the genome (present on 20/23 chromosomes counting the sex chromosomes) are transcribed by RNA pol II in association with MYC:MAX and the recruitment of the HAT complex to the promoter. The mRNAs encoding the RPs are processed and transported to the cytoplasm for translation. RNA splicing of these transcripts also produces the scaRNAs and snoRNAs later needed for the formation of diverse heterogeneous ribonucleoprotein (hnRNP) complexes, C/D snoRNPs, and H/ACA snoRNPs that function in mRNA splicing and rRNA modification/maturation. Alternative splicing of RP transcripts also produces “pseudogenes” that regulate the expression/accumulation of RPs. Newly translated RPs are then actively imported from the cytoplasm to the nucleolus and nucleoplasm where they are incorporated into the assembling ribosome. This process requires the presence of diverse chaperone proteins that serve several functions: (1) Protect the RPs from degradation, (2) facilitate their active nuclear import, and (3) assume the correct incorporation of the RPs into the maturing ribosome subunits.  .

The initial 47S pre-rRNA transcript maintains a secondary structure at the newly synthesized 5′-end that acts as a platform for the binding and association of an initial set of RPs forming the 90S RNP. As the 5’ portion of the pre-rRNA contains what will become 18S rRNA of the 40S subunit, the RPs associating with the 5′-end are small subunit RPs or RPSs. Two of the first RPs to associate with the pre-rRNA at the 5′-end are RPS7 and RPS24, which are required to initiate processing and cleavage of the pre-rRNA at the 5′-external transcribed spacer (ETS). The initial binding of the RPSs and the processing at the 5’-ETS, as well as modifications of the rRNA by ribose methylases (C/D box snoRNPs) and polyuridylases (H/ACA box snoRNPs), energetically favors an rRNA structure that forms a platform for the next round of RPs to associate. At the same time that processing of the 5′-end of the 18S rRNA is being conducted, association of RPS17 and RPS19 facilitate the processing and cleavage at the 3′-end of the 18S rRNA within the internal transcribed sequence (ITS1), liberating the assembling 40S subunit from the 60S subunit. Low levels of RPS7 or RPS24 block or retard rRNA processing, resulting in failed maturation of the 5′-end of the 18S rRNA. Similarly, depletion of RPS17 or RPS19 result in failed processing of the 3’-end of the 18S rRNA. Virtually the same process occurs for the formation of the maturing 5.8S and 28S with the ITS2 (between 5.8S and 28S sequences). At this point, the associating RPs are RPLs as they will become part of the 60S subunit. The process, marked by rounds of rRNA processing and folding and rounds of RP association, continues to occur until the last of the RPs for each subunit are finally incorporated in the cytoplasm. Due to the ability of RNA to assume diverse energy structures at equilibrium, diverse modes of RNP assembly can be occurring simultaneously, including the synthesis of kinetically dead-end products that are aborted and targeted for degradation and recycling by the TRAMP4/5 complex. The enzymes and proteins that modify and cleave the 47S rRNA are numerous and the process complex;

Several lines of evidence indicate that the main purpose of the RPs is to stabilize rRNA folding and structure, thus assisting in processing; 

(i) the RPs are RNA-binding proteins that are dependent on RNA structure and conformation, 
(ii) there are very few protein–protein interactions between the RPs, 
(iii) while many of the RPs possess a tail that inserts into the ribosome core, the core is, proteically speaking, mostly hollow, with the exception of the rRNA species; and the RP tails apparently do not interact with the mRNA substrate that will later occupy the core. 

In addition to the RPs incorporated into the ribosome and the rRNA modifying proteins, a number of proteins associate with the maturing ribosome in the nucleus to ensure that the ribosome does not prematurely assemble, including the Shwachman–Diamond Syndrome associated protein, SBDS, and eukaryotic initiation factor 6 (eIF6). Once exported into the cytoplasm, these chaperons disassociate from the ribosomal subunits, allowing for ribosomal subunit assembly and the association of mRNA and translation initiation factors (IFs) to form the PIC. Final steps involving the eIF2, eIF4, and eIF5 complexes result in mature ribosomes and the initiation of protein synthesis. Alteration at any step of this process can have mild to dire consequences depending on the defect and its penetrance. Tissues, which have the greatest rates of proliferation and turn-over, such as the skin and hematopoietic precursors, especially of the erythroid lineage, are often the most affected.

Many RPs also have other trans-regulatory functions. Processing of the 60S subunit requires the incorporation of the 5S rRNA, which initially associates with RPL5 and RPL11 to form the 5S RNP. Under the conditions of RP haploinsufficiency or disturbances in ribosome biogenesis as a result of altered signaling or stress, the process of pre-rRNA maturation is arrested, resulting in an increasing pool of unassimilated RPs. The accumulation of free RPs, namely the 5S RNP, results in the inhibition of protein synthesis and cell cycle arrest through the activation of p53. Other than enhanced p53 expression, other mechanisms may explain the pro-apoptotic and poor proliferative state observed, following the loss or reduced expression of RPs. Loss of RPL5 or RPL11 was shown to lead to reduced cyclin expression, and therefore, reduced proliferation. Additionally, cells deficient in RPS19 and RPL11 were demonstrated to be predisposed to oxidative stress. In addition, an innate immune component has been observed. RP loss/reduction enhances the expression of innate immune genes including interferon and TNFα, which is known to contribute to the hematopoietic failure in RPS19 deficiency, likely through IRES-mediated translation.

Ribosome biogenesis in replicating cells: integration of experiment and theory 2017 Oct 1 4
Ribosomes—the primary macromolecular machines responsible for translating the genetic code into proteins—are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. In Escherichia coli, ribosomes account for approximately one fourth of the cellular dry mass and the majority of the total RNA. It can be tempting, then, to think of the bacterial cell as a finely tuned machine for building ribosomes. Their role in protein synthesis involves them (either directly or indirectly) in essentially every process within the cell.

Ribosome production is tightly regulated by the cell. This is no small feat, considering that each 70S ribosome involves the coordinated transcription, translation, folding, and hierarchical assembly of three strands of rRNA and over four dozen proteins, all within the heterogeneous, crowded intracellular space.. Work on the 30S small subunit (SSU), which is largely responsible for recognizing and decoding mRNA, showed that assembly nucleates with the folding of the so called five-way junction in the 16S rRNA of the SSU (residues 27–45 and 394–554 in E. coli), and then proceeds through the hierarchical association of sets of ribosomal proteins, each progressively folding and stabilizing the rRNA's growing tertiary structure. Interestingly, a number of in vitro studies have observed this process proceeding over timescales on the order of the cell cycle or longer, while in vivo it can take just a few minutes. 

Ribosomal RNA is transcribed from seven operons interspersed throughout the E. coli genome, and many of the intermediate structures along the assembly pathways can exist in very few copies due to the rapid binding of additional proteins. The kinetic model of ribosome biogenesis includes seven ribosomal RNA operons which code for the 16S rRNA and nine operons coding for the 18 ribosomal proteins (r-protein), which along with the 16S rRNA, compose the 30S small subunit of the ribosome.

Driving ribosome assembly
The ribosome is a complex molecular machine that is composed of a small 40S and large 60S subunit. Despite their conserved molecular function, eukaryotic and prokaryotic ribosomal subunits differ significantly in size and complexity. Ribosome biogenesis faces the challenge to coordinate the processing and modification of ribosomal RNA (rRNA) with its correct structural assembly with ribosomal proteins (RP). Furthermore, this process has to be regulated according to the cellular environment, hence ribosome biogenesis is tightly coupled to growth rate: actively dividing cells, including cancer cells, depend on active ribosome biogenesis, whereas arrested or starving cells halt the production of new ribosomal subunits. Due to its easy experimental accessibility by genetic, biochemical, and cell biological methods, S. cerevisiae represents a suitable eukaryotic model organism to study ribosome assembly and the function of non-ribosomal factors. Over the past 20 years, it has been shown that a large number of non-ribosomal factors (N 200) and snoRNAs (75) are involved in ribosome assembly.  pre-ribosomal particles along their maturation path are also involved. The current challenge is to identify direct interaction partners of individual proteins and obtain structural information of single proteins and pre-ribosomal particles. 1

In eukaryotic cells, ribosomes are preassembled in the nucleus and exported to the cytoplasm where they undergo final maturation. This involves the release of trans-acting shuttling factors, transport factors, incorporation of the remaining ribosomal proteins, and final rRNA processing steps. Recent work, especially on the large (60S) ribosomal subunit, has made it abundantly clear that the 60S subunit is exported from the nucleus in a functionally inactive state. Its arrival in the cytoplasm triggers events that render it translationally competent. Here we focus on these cytoplasmic maturation events and speculate about why eukaryotic cells have evolved such an elaborate pathway of maturation.

The biogenesis of ribosomal subunits –“state of the art”
In all living cells, the ribosome is responsible for the final step of decoding genetic information into proteins. This universal “translating apparatus” comprises two subunits, each of which is a complex assemblage of RNA and proteins (. The two subunits display a distinct division of labor: the small 40S subunit (30S in prokaryotes) is responsible for decoding whereas the large 60S subunit (50S in prokaryotes) carries out the chemistry of polypeptide synthesis.  How do cells assemble such an intricate machine and ensure that it functions faithfully in the critical role of decoding a cell’s genome?  2

Ribosome biogenesis begins with transcription of the pre-rRNA, which undergoes co-transcriptional folding, modification, and assembly with ribosomal proteins (r-proteins) to form the two subunits. The assembly of ribosomal subunits in bacteria appears to require few (<25) trans-acting factors. By contrast, eukaryotic ribosome assembly is a complicated process that requires the concerted efforts of all three RNA polymerases and >200 trans-acting factors, that aid the assembly, maturation and intracellular transport of ribosomal subunits.

Eukaryotic ribosomes are initially assembled in the nucleolus, the site of rRNA transcription. Although the nascent pre-ribosomal particles released from the nucleolus appear to be largely preassembled, they require additional maturation steps in the nucleoplasm and/or cytoplasm. The pre-ribosome, the 90S particle, is subsequently processed to yield smaller 66S and 43S particles, the precursors to the mature 60S and 40S subunits, respectively. These particles contain pre-rRNA, r-proteins, and numerous trans-acting factors. There are multiple trans-acting factors.

Amino Esters and Ribosomes
DNA’s four-character alphabet is used to compose the larger twenty-character alphabet of alpha-amino acids (α-amino acids). Life needs this collection of twenty building blocks, each distinct, to make a protein. These building blocks must react with each other to form specific chemical connections called peptide bonds. Chemists have learned to use this reaction to make polymers like nylon, for which they used H2N- (CH2)6-COOH molecules as the specific building blocks. The reaction occurs without much guidance because NH2 has no option but to react with COOH. It’s much more complicated for proteins, however, since α-amino acids have twenty different side chains (called “R groups”; attached to their backbones. Each protein is a polymer, a chain made of many subunits linked together like nylon, but composed of amino acids. But the amino acid R groups pose a serious problem for protein synthesis, because they can react favorably with both themselves and the COOH and NH2 groups of the other α-amino acids. The desired peptide reactions, on the other hand, are usually unfavorable, requiring a positive change in free energy. All the other viable side reactions will interfere with the formation of a protein polymer. So how does life get around this severe competition problem? Life relies on a chemical trick often used in synthetic chemistry: derivatization. Ribosomes are large multimolecular machines that synthesize proteins from amino acids in living cells. But before going to ribosomes, each α-amino acid is converted into an amino ester, a process called “derivatization,” and attached to a “transfer RNA” (tRNA) by an enzyme called a t-RNA synthetase. There are distinct tRNAs and tRNA synthetases for each amino acid. Competition from energetically more favorable R-with-R or even R-with-NH2 or R-with-COOH reactions would be fatal to protein synthesis if it were not for the ribosome. Here’s what happens during the process of translation, as α-amino acids get attached to their specific t-RNA by their specific t-RNA synthetases. In a very elegant and ingenious process, amino esters are first phosphorylated by ATP and then, via a trans-esterification reaction, a t-RNA linked amino ester is formed. To ensure the desirable NH2-with-COOH reaction takes place, the amino acids are first esterified (which makes the chemical bond easier to form), then brought together by the mechanical hands of a ribosome, holding them in the correct position to prevent competing R reactions from taking place, and providing the necessary energy for the bond to form. Again, this ribosome-driven reaction does not seem to be an advantage that life could acquire little by little, by trial and error. Chemically, it is impossible to produce a functional protein without ribosomes that have already solved the competing reaction problem, or without the collection of twenty specific tRNAs and tRNA synthetases that would feed it with amino esters. As in so many other cases with the cell and its code, if this need is not foreseen and planned for, there will be no cell at all.

The ribosome is a vital machine in every cell. It is stunning in the details required to make this machine. I like the insight gained through new research. When it is made "It doesn't just go plop". It is a multistep process requiring perfect precision and timing.
Not something unintelligent processes can produce!!
Again glory to God for life!!

"The synthesis of ribosomes is therefore an extremely complex, multistep process, which includes both assembly and maturation stages."

"The "emergence" of the 40S subunit entails an ordered series of reactions in which the outer shell of the 90S particle is progressively dissociated from the 40S. "It doesn't just go plop," Beckmann remarks. The process is actually reminiscent of the molting of an insect—shedding of the integument takes place layer by layer. "It's rather like those Russian dolls. When you open one, you find a smaller one nestled inside," says Beckmann

https://phys.org/news/2020-09-ribosomes-russian-dolls.html?fbclid=IwAR0YcGlXqObFJAO3_RGxFNUIx271iXe43ljT4gPx-AhBqq2UxIWjbPWbvxc

Ribosome Biogenesis in Eukaryotes
Eukaryotic ribosomes are the 80S ribosomes, consisting of 60S large subunit and 40S small subunit. The large subunit consists of three types of rRNAs (25S in plants or 28S in mammals, 5.8S, and 5S) and about 47 ribosomal proteins. The small subunit consists of one rRNA (18S) and about 33 ribosomal proteins.

Ribosome Biogenesis in Prokaryotes
70S ribosomes, which consist of 50S large and 30S small subunits, are prokaryotic ribosomes. The large subunit consists of two types of rRNAs (23S and 5S) and 33 ribosomal proteins. The small subunit consists of only 16S rRNA and 20 ribosomal proteins.

52 genes of the prokaryotic genome encode the ribosomal proteins. They belong to 20 different operons. Transcription of the ribosome gene operons occurs in the cytoplasm. The synthesis of ribosomal proteins as well as the assembly of large and small subunits of the ribosome occurs in the cytoplasm in prokaryotes.

153 proteins (including 91 non-ribosomal proteins) that are required for ribosome synthesis in human cells. 5

Ribosome assembly coming into focus   22 November 2018 6
Protein synthesis in eukaryotes is carried out by the ribosome, which is a large RNA–protein complex consisting of a small and a large subunit. During protein synthesis, decoding of mRNA by the small subunit is coupled with peptide- bond formation by the large subunit. In the model organism Saccharomyces cerevisiae, the small subunit (40S) comprises 33 ribosomal proteins and the 18S ribosomal RNA (rRNA), whereas the large subunit (60S) comprises 46 ribosomal proteins and 3 rRNAs (25S, 5.8S and 5S rRNA). Whereas the ribosomal catalytic centres — the decoding site in the small subunit and the peptidyl transferase centre (PTC) in the large subunit — and other ribosomal functional modules and key architectural features, such as the central pseudoknot in the small subunit and the central protuberance, GTPase activating centre (GAC), P0 stalk and polypeptide exit tunnel (PET) in the large subunit, are evolutionarily conserved, eukaryotic ribosomes contain many additional RNA extensions and proteins. The assembly and thus availability of eukaryotic ribosomal subunits is intimately linked to nutrient availability, stress and the cell cycle. Approximately 200 non- ribosomal factors, including proteins, protein complexes and small nucleolar ribonucleoproteins (snoRNPs), are required for the assembly of the small and large ribosomal subunits. The maturation of pre- rRNAs for both subunits requires endonucleolytic and exonucleolytic cleavage (Fig. 1). 


Fig. 2 Pre-rrNa processing in yeast. 
Consecutive pre-ribosomal RNA (pre-rRNA) processing stages produce rRNA intermediates through endonucleolytic (cleaves nucleic acids in half by hydrolyzing (break down (a compound) by chemical reaction with water) the bonds between nucleotides) and exonucleolytic (cleaves nucleic acid by the removal of single nucleotides from the end of the chain.) removal of internal transcribed spacers (ITSs) and external transcribed spacers (ETSs). Precursors of mature rRNAs destined for the small ribosomal subunit are shown in green and those for the large ribosomal subunit in pink. Processing and assembly begin in the nucleolus, continue in the nucleoplasm and are completed in the cytoplasm. Sites in pre-rRNAs at which endonucleases cleave (A0, A1, A2, A3 and C2) or exonucleases halt (B1) are highlighted in red at the step at which they occur. Each precursor RNA is designated by its size (assayed by velocity sedimentation on sucrose gradients) and by the site processed to generate that intermediate (for example, 27SA2). Each of these processing steps occurs within the indicated pre- ribosomal ribonucleoprotein particle. In rapidly growing yeast cells, processing and assembly occur mostly co- transcriptionally , with cleavage at the A0, A1 and A2 sites occurring in nascent pre- rRNAs, indicated by production of the 20S pre- rRNA (shown on the left). By contrast, in slowly growing yeast cells, processing occurs post- transcriptionally , with processing of the full- length 35S pre- rRNA (top) by cleavage at the A3 site to generate the 23S pre- rRNA and then at the A0, A1 and A2 sites (dashed arrow on the right). SSU processome, small- subunit processome.

Distinct stages of this process take place first in the nucleolus, then in the nucleus and finally in the cytoplasm. Following a general overview of smallsubunit assembly (Fig. 2),



we provide a cryo- electron microscopy (cryo- EM) depiction of a nucleolar smallsubunit intermediate (Fig. 3). 


Fig. 3 | cryo- electron microscopy structure of a nucleolar small- subunit precursor. a–c | 
Three views of the smallsubunit processome (PDB ID: 5WLC), highlighting the overall architecture (part a), organization of long peptide extensions that bridge distant regions in the particle (part b) and the interaction between pre-18S ribosomal RNA (rRNA), 5ʹ external transcribed spacer (ETS) and U3 small nucleolar RNA (U3 snoRNA) (part c). d | Schematic representation of secondary structures of pre-18S rRNA and U3 snoRNA. The four domains of the pre-18S rRNA (5ʹ, central, 3ʹ major and 3ʹ minor) are colour- coded. Base pairing of the U3 snoRNA 3ʹ hinge and 5ʹ hinge with the 5ʹ ETS, and of the U3 snoRNA Box A and Box Aʹ sequences with pre-18S rRNA are indicated. Protein complexes are outlined as transparent surfaces in parts b and c.

Similarly to the illustration of the small subunit in Fig. 2, the arrival and departure of ribosome assembly factors for the large subunit are illustrated with structures of pre- ribosomal particles in Fig. 4 and serve as a general guide for ribosome assembly. 


Fig. 4 | assembly of the large ribosomal subunit. 
Consecutive stages in the maturation of the large ribosomal subunit (60S) are shown, from the earliest stages in the nucleolus, through stages in the nucleoplasm and finally in the cytoplasm. Large- subunit-specific portions of ribosomal DNA (rDNA) are depicted with colour- coding of the 5.8S ribosomal RNA (rRNA), the internal transcribed spacer 2 (ITS2), the 25S rRNA domains I–VI and the 3ʹ external transcribed spacer (3ʹ ETS). Six assembly intermediates for which cryoelectron microscopy (cryo- EM) structures have been determined are shown: state 1 or state A (state 1/A), state 2/B, state E, Nog2, Rix1–Mdn1 and Nmd3 particles. Pre- rRNA intermediates present in each particle are indicated in square brackets, and rRNA domains that have assembled into stable visible domains are depicted using the same colours of the rDNA. Note that some of the different particles contain the same pre- rRNAs but differ in structure and protein content (for example, state 1/A and state 2/B). There are likely additional assembly intermediates to be discovered. The association and dissociation of assembly factors is shown. Assembly factors for which structural information is available are depicted in cartoon form; those for which no structures are known are indicated with text only. The earliest preribosomal particles present before state 1/A particles are formed cotranscriptionally and have not been visualized by electron microscopy. In the state 1/A and state 2/B particles, 25S rRNA domains I, II and VI and the 5.8S rRNA and ITS2 have begun to form and become stable, visible conformations. The transition from state 2/B to states C and D (which are not shown as particles), and then to state E, involves assembly of domains III, IV and V and includes early steps in the formation of the peptidyl transferase centre and polypeptide exit tunnel functional centres. Major structural remodelling occurs to form Nog2 particles, which translocate from the nucleolus to the nucleoplasm, where additional restructuring as well as quality control checkpoints are carried out to prepare particles for nuclear export. Upon entry into the cytoplasm, the remaining assembly factors are released, as the assembly and surveillance of functional centres is completed. The ‘wiggling’ signs highlight components that are flexible. NPC, nuclear pore complex.

The yeast nomenclature for ribosomal proteins is used here. Nucleolar ribosome assembly is characterized by the co-transcriptional association of ribosome assembly factors with nascent pre-rRNA. Reduction of conformational freedom of the nascent pre-rRNA aids the formation of subdomains for both the small ribosomal subunit (Fig. 3) and the large ribosomal subunit (Fig. 5). 


Fig. 5 |cryo- electron microscopy structures of large- subunit precursors. 
a | Architecture of an early nucleolar pre-60S particle known as state 2 or state B (state 2/B; PDB ID: 6C0F), which includes the folded domains I, II and VI of pre-25S ribosomal RNA (rRNA) and the pre-5.8S rRNA. 
b | Architecture of a nucleolar pre-60S particle known as state E. Note that domains III and V, and expansion segment 27 (ES27) of domain IV of the pre-25S rRNA , are now folded and visible. 
c | Architecture of the late nucleolar Nog2 particle. Here, domain IV is folded and visible with the 5S rRNA. Components already present in the previous particle are shown as transparent. 
d–f | Diagrams of the large- subunit rRNA secondary structure, showing the successive folding and stabilization of subdomains. Parts d–f depict rRNA organization of state 2/B, state E and Nog2 particles, respectively.
Stably ordered RNAs (solid lines), largely ordered RNAs (dashed lines) and disordered RNAs (in light grey) are indicated with the same colour- coding as in parts a–c. CTD, carboxy- terminal domain; ITS2, internal transcribed spacer 2; NTD, amino- terminal domain.

During nuclear ribosome assembly, the relative orientations of these subdomains are already closer to the mature conformations in the small and large subunits, but the subdomains still undergo extensive remodelling (Fig. 6).


Fig. 6 | Structural changes occurring in nucleolar and nuclear ribosomal precursors. 
a,b | Rearrangement of the relative orientations of pre-18S rRNA domains during the transition from the small- subunit processome  to the cytoplasmic pre-40S particle. The 5′, central, 3′ major and 3′ minor domains and the U3 small nucleolar RNA (U3) are indicated. These conformational changes are indicated by arrows in part a. 
c–e | Maturation of large- subunit particles near the Nog1 binding site in the nucleolar pre-60S particle (also known as state E; PDB ID: 6ELZ, manual building), the nucleolar Nog2 particle and the nuclear Rix1–Mdn1 particle (PDB ID: 5JCS, manual building). In the transition from state E to Nog2 particles, the release of assembly factors Spb1, Noc3, Nip7 and Nop2 enables maturation of the polypeptide exit tunnel and stable docking of the 5S ribonucleoprotein (RNP) through Rpf2 and Rrs1. Subsequent formation of the nucleoplasmic Rix1–Mdn1 particle involves release of Rpf2 and Rrs1 to destabilize the pre- rotated state of the central protuberance and assembly of Sda1, the Ipi1−Rix1−Ipi3 complex and Mdn1 to stabilize the rotated state of the central protuberance. CTD, carboxy- terminal domain; NTD, amino- terminal domain.

Lastly, in the cytoplasm, final adjustments and steps of quality control are employed to test the functionality of both subunits for protein synthesis. Ribosome assembly factors associated with precursors of the small ribosomal subunit are chronologically listed in Supplementary Table 1, and those associated with precursors of the large ribosomal subunit are listed in Supplementary Table 2.

There are four key concepts of ribosome assembly: the systematic reduction of conformational freedom of pre- rRNA during early nucleolar stages; the chronology of assembly factor binding, which is enforced by molecular mimicry and molecular switches to prevent premature folding states or processing steps and enable timely progress of assembly; the irreversibility of key checkpoints, which is dependent on energy consumption and RNA- processing enzymes that can bring about structural changes; and the importance of structural and functional proofreading of functional centres of both ribosomal subunits.

Nucleolar assembly
In almost all eukaryotes, the small- subunit rRNA sequences are located close to the 5ʹ end and the largesubunit rRNA sequences are located close to the 3ʹ end of the precursor rRNA. In S. cerevisiae, RNA polymerase I
(Pol I) transcribes a 35S rRNA precursor, which, in addition to external and internal transcribed spacers — the 5ʹ external transcribed spacer (5ʹ ETS), 3ʹ ETS, internal transcribed spacer 1 (ITS1) and ITS2 — contains the 18S rRNA for the small subunit and the 25S and 5.8S rRNAs for the large subunit (Fig. 1). Pol III separately transcribes the 5S rRNA, which is later integrated into the large subunit.

Co-transcriptional pre-rRNA processing. 
Recent structural studies have elucidated how transcription is catalysed by Pol I, which is a 14-subunit complex that transcribes rRNA with the assistance of a dedicated set of initiation factors. Although it is known that either Pol I or Pol II can be used to transcribe rRNA, Pol I is inactivated through dimerization following glucose depletion, thereby providing a direct link between nutrient availability and rRNA synthesis. Processing of pre- rRNA can occur either cotranscriptionally or post-transcriptionally. A 35S pre-rRNA species can be synthesized first and the entire precursor for both ribosomal subunits (then referred to as 37S rRNA) was present in 90S particles. In rapidly growing cells, the majority of processing (~70%) occurs co-transcriptionally. Co-incident with transcription, rRNA undergoes covalent modifications, most of which are clustered in functionally important domains and are thought to fine-tune rRNA structure or function. Different classes of factors are associated with the modification of prerRNA. Among the RNA-containing classes, these include snoRNPs, which can catalyse either pseudouridylation (H/ACA snoRNPs) or 2ʹ-O-ribose methylation (box C/D snoRNPs) of pre-rRNA. Both H/ACA snoRNPs and box C/D snoRNPs are guided by snoRNAs, which contain sequence elements that base-pair with the target rRNA, and structural motifs for binding dedicated protein cofactors. Box C/D- associated proteins include the methyltransferase Nop1, the Nop56–Nop58 heterodimer and Snu13; H/ACA- associated proteins include the pseudouridine synthase Cbf5 and Gar1, Nop10 and Nhp2. Two other snoRNPs that have central roles in eukaryotic ribosome assembly are the U3 snoRNP and RNase MRP (mitochondrial RNA processing). The box C/D family U3 snoRNP is a key structural organizer for the assembly of the small subunit and for co-transcriptional cleavage of the small-subunit pre-rRNA at sites A0, A1 and A2 (Fig. 3c,d), but this does not result in 2ʹ-O- ribose methylation. By contrast, RNase MRP catalyses the cleavage of site A3, a site that is also associated with post- transcriptional cleavage (Fig. 1). In addition to RNA-mediated RNA modifications, 19 RNA helicases, including DEAD-box and DEAHbox helicases, have been implicated in yeast ribosome assembly. Seven of these factors (Dbp4, Dbp8, Dhr1, Dhr2, Fal1, Rok1 and Rrp3) are involved in small- subunit assembly, nine helicases (Dbp2, Dbp3, Dbp6, Dbp7, Dbp9, Dbp10, Drs1, Mak5 and Spb4) are involved in large-subunit assembly and three (Has1, Mtr4 and Prp43) have been implicated in the assembly of both subunits. Additional energyconsuming enzymes include GTPases such as Bms1, Nog1, Nog2, Nug1, Lsg1 and Efl1; ATPases such as Rio1, Rio2 and Fap7; and AAA- ATPases (Mdn1, Drg1 and Rix7). The role of these factors is to drive ribosome assembly in a unidirectional manner. Many of the assembly factors have no predicted enzymatic functions but contain RNA- binding motifs and can be seen in the structures to bind to rRNA. Finally, the assembly of a number of ribosomal proteins into ribosome intermediates is aided by dedicated chaperones, which enable their co-translational folding, escort them into the nucleus or help insert them into pre-ribosomal particles undergoing assembly. The connection between the terminal structures
observed in Miller spreads and the association of prerRNA with small- subunit ribosome assembly factors has long remained unclear. The terminal ball structures present in Miller spreads were originally hypothesized to contain pre-rRNA, but later studies identified them as rRNA- processing complexes that contain the 5ʹ ETS in Xenopus laevis and require U3 snoRNA. The identification of the small- subunit processome (SSU processome) as a U3 snoRNA- associated pre-ribosomal particle that contains many ribosome assembly factors provided the first evidence linking the previously observed terminal structures to a defined macromolecular complex. In another study, a similar particle containing smallsubunit, but lacking large- subunit, biogenesis factors was described, which was termed the 90S pre- ribosome as it was assumed to contain a 35S pre-rRNA. As all current data suggest that both particles are indeed the SSU processome (as they do not contain the 35S prerRNA and lack large-subunit assembly factors), we refer to this particle as the SSU processome.

Nucleolar assembly of the small subunit. 
Early stages of nucleolar ribosome assembly occur cotranscriptionally, and the temporal association of many ribosome assembly factors with pre-ribosomal particles occurs dynamically at 100–200 ribosomal DNA (rDNA) loci.  During the formation of the SSU processome, prerRNA undergoes chemical modifications and the four domains of the 18S rRNA (the 5ʹ, central, 3ʹ major and 3ʹ minor domains) begin to be formed. These subdomains contain independent secondary structure elements, which in the mature small subunit form the 3D structure of the 18S rRNA (Supplementary Figure 2). Importantly, both RNA and protein factors are involved in the reduction of conformational freedom of prerRNA. This dual involvement is used to orient the four subdomains in a conformation that precludes their premature folding while allowing each domain to be assembled separately in an encapsulated environment. This encapsulation is conceptually different from largesubunit maturation, which occurs in a modular fashion, where subdomains are bound by more-isolated assembly factors. The formation of the 5ʹ ETS pre-ribosomal particle provides the first scaffold on which the 5ʹ ETS; the protein complexes UtpA, UtpB and Mpp10; the U3 snoRNP (an important RNA chaperone complex); and smaller individual proteins provide an architectural support for the SSU subdomains, which are formed subsequently (Fig. 3a,b). U3 snoRNA has a central role in the formation of the SSU processome, as it base- pairs with and therefore rigidifies regions in both the 5ʹ ETS (with its 5ʹ and 3ʹ hinges) and the pre-18S RNAs (with its Box A and Box Aʹ) (Fig. 3c,d). The close proximity of these binding sites within the 5ʹ region of U3 snoRNA provides a crucial spatial constraint that dictates the topology of the maturing particle. Similarly, several of the early multimodal binding proteins (Utp11, Sas10, Mpp10 and Fcf2) confine pre-rRNA domains within the particle by binding to either protein or RNA elements. In addition, many factors have transient roles in the biogenesis of early small- subunit particles and may be present co-transcriptionally only before the cleavage at site A2. These include both small RNAs, such as U14, snR10 and snR30, and proteins that have been associated with each of the 18S rRNA subdomains. In comparison with the more stably associated assembly factors, which form part of the mature SSU processome, the roles of these more transiently bound factors are currently less well understood (Fig. 2). RNA cleavage events represent irreversible steps that separate pre-rRNAs from each other and from ITSs or ETSs. During co-transcriptional pre-rRNA processing, cleavage occurs at sites A0, A1 and A2, thereby liberating a 20S pre- rRNA precursor; by contrast, during post-transcriptional processing, RNase MRP cleaves at site A3 to generate a 23S precursor, which is processed further (Fig. 1). The function of the 23S rRNA is not fully clear, as it has also been associated with aberrant pre-rRNA processing in response to depletion of smallsubunit assembly factors and large-subunit ribosomal proteins. Whereas recent evidence suggests that Utp24 is the nuclease responsible for cleavage at sites A1 and A2, the nuclease responsible for cleavage at site A0 remains to be identified. Recently, cryo- EM structures of the SSU processome have been determined that illustrate its architecture and near- atomic structure. The near-atomic structures together with crystallography data and protein–protein interaction data now provide a molecular snapshot of the earliest stable precursor of the small ribosomal subunit. It is likely that the SSU processome is an intermediate that forms under physiological conditions. The yeast SSU processome provides an encapsulated environment for the rRNA subdomains of the small subunit. The base of the particle is formed around the 5ʹ ETS and the protein complexes UtpA and UtpB, which are related and act as molecular chaperones at the base and side of the particle, respectively (Fig. 3). The top of the particle contains the 5ʹ, central and 3ʹ domains of the rRNA, each of which is housed in a separate region of the SSU processome. The interconnectivity of many proteins containing long peptide extensions, such as Mpp10, Utp11 and Sas10 (Fig. 3b), and the base pairing of U3 snoRNA to both the 5ʹ ETS and 18S rRNA (Fig. 3c,d) provide further structural support for the particle. Ribosome assembly factors that are stably associated with a subdomain of the pre-18S rRNA predominantly act as local stabilizers of RNA elements. Both helical- repeat-containing proteins (Nop14, Noc4, Rrp5, Utp10 and Utp20) and several enzymes, such as the methyltransferase Emg1, the acetyltransferase– helicase Kre33 and the GTPase Bms1, are located in the outer regions of the SSU processome. Whereas the roles of the helical- repeatcontaining proteins are clearly structural, the temporal order in which the enzymes act on the encapsulated pre-18S rRNA remains to be determined. A key structural role of the SSU processome is chaperoning the assembly of each of the subdomains of the 18SrRNA while preventing the premature formation of the central pseudoknot. However, in all currently available structures, the SSU processome contains a highly intertwined pre-18S rRNA, which needs to be released from the SSU processome for further maturation (Fig. 2). To achieve this, RNA helicases such as Dhr1, which has a role in U3 snoRNA unwinding, are necessary to liberate the 18S precursor from U3 snoRNA. Similarly, cleavage at sites A1 and A2 by Utp24 is necessary but not possible in the available structures because the active site of Utp24 is inaccessible. Lastly, the processing of the SSU processome, in particular the removal of the 5ʹ ETS, requires the exosome and exosome- interacting proteins such as Utp18, Sas10 and Lcp5.

Assembly of the large ribosomal subunit
The structure of the large subunit is more elaborate than that of the small subunit; consequently, its assembly follows a more complex pathway. The 25S rRNA in the large subunit consists of six conserved domains of secondary and tertiary structure, namely (5ʹ to 3ʹ) domains I–VI, which are more intertwined with each other than are the 18S rRNA domains in the small subunit (Supplementary Figure 2). The solvent- exposed surface of the large subunit includes domains I and II and the 5.8S rRNA, whereas the subunit interface contains functional centres that include domains IV and V. Domains III and IV bridge one edge of the solvent- exposed surface and subunit interfaces; domain III also links RNA domains at the bottom of the subunit. The 5.8S rRNA lies between domains I and III, and 5S rRNA is docked on top of domains II and V (Supplementary Figure 2). Because we lack cryo- EM structures for the earliest stages of 60S subunit assembly in the nucleolus, our understanding is so far based only on biochemical and genetic studies of pre- rRNA processing and of the assembly factors that participate in initial packaging of pre-60S ribosomal ribonucleoproteins (rRNPs). By contrast, cryo- EM snapshots of a number of assembly intermediates following the earliest nucleolar stages of assembly support more detailed models of the later stages of large- subunit biogenesis (Figs 4, 5). The assembly of the large subunit begins co- transcriptionally, with the covalent modification of pre- rRNA, which is guided by snoRNPs. At the same time, folding of the long, flexible pre-rRNA into more compact and stable conformations and cleavage and processing of the ITS1 and 3ʹ ETS (Fig. 1) occur. The solvent- exposed surface of the large subunit is formed first by folding of the 5.8S rRNA with 25S domains I and II (seen in state 1 or state A (state 1/A)) and then with domain VI (state 2/B) into stable structures (Fig. 5a,d). The intervening domains III, IV and V are initially flexible and bound by assembly factors. As the 25S rRNA domains III, IV and V become structurally visible and join the already folded domains I, II and VI (Fig. 5b,e), initial stages of construction of the functional centres of the large subunit (the PTC and PET) become evident (state E). During the following transition from the nucleolus to the nucleoplasm (Figs 4; 5c,f), numerous protein exchanges enable other major remodelling events, leading to export from the nucleus. Then, in the cytoplasm, pre-60S ribosomes undergo final stages of maturation, including the removal of remaining assembly factors, assembly of the last few ribosomal proteins and test-driving of functional centres.

Nucleolar stages of large- subunit assembly. 
Among the assembly factors that participate in the earliest cotranscriptional stages of large-subunit assembly are several proteins that contain multiple RNA-binding domains or α-helical repeats, such as Rrp5, Mak21,
Noc2 and Nop4. These proteins might enable rRNA compaction by forming rigid scaffolds to direct and stabilize RNA folding. In Miller spreads, the terminal ball structures corresponding to pre-ribosomal particles have a more loosened structure in mutants lacking these assembly factors. The early assembly factors Npa1, Npa2, Rsa3 and Nop8 and the RNA helicase Dbp6 form a stable complex that also may serve a structural function. Six other RNA helicases (Dbp2, Dbp3, Dbp7, Dbp9, Mak5 and Prp43) are also required for these very early assembly stages, most likely to remodel RNA or RNPs. However, the binding sites and precise targets of these enzymes have not yet been defined. Interestingly, cleavage at both the A2 and A3 sites in the ITS1 is linked to transcription or processing of sequences several kilobases away from those sites. Cotranscriptional cleavage at the A2 site occurs only once Pol I reaches ~1.2–1.5 kb downstream of it, in the domain I and domain II portion of 25S rDNA sequences, and cleavage at the A3 site (near what will become the 5ʹ end of 27SB pre- rRNA) occurs only upon termination of transcription and processing of the 3ʹ ETS several kilobases downstream. Exactly how these cleavages are coupled to downstream events is unclear, but large-scale as well as local folding of RNA mediated by protein binding might be necessary to create proper substrates for cleavage. For example, binding of Rrp5 near pre- rRNA processing sites in both the SSU processome (the A2 site) and pre-60S particles (the A3 site) may regulate the timing of cleavage at these sites and coordinate assembly of both subunits. The amino- terminal domain half of Rrp5, which contains multiple RNAbinding motifs that bind close to the A3 cleavage site in ITS1, is necessary for A3 cleavage. The carboxy- terminal half of Rrp5, which contains additional RNA- binding motifs, crosslinks near the A2 cleavage site in ITS1 and is necessary for A2 cleavage. Thus, proper folding of ITS1 sequences as well as domain I and domain II bound to the amino- terminal domain of Rrp5 may be necessary to signal successful initiation of assembly of the large subunit before the SSU processome can be separated from pre-60S particles by cleavage at the A2 site. Likewise, 3D proximity of the 5ʹ end and 3ʹ end of the 27SB pre- rRNA suggests that their processing may be coordinated, perhaps through protein–protein or protein–RNA interactions of Rrp5 and ribosomal protein L3, which bind to these termini66,92. Cryo- EM structures103–105 begin to reveal how the rRNA domains of early nucleolar pre-60S subunits are held together: the assembly factors and ribosomal proteins form bridges within and between each rRNA domain. For example, in state 2/B particles, three assembly factors (Nop15, Cic1 and Rlp7) bind to the ITS2 and five others (Nop7, the Erb1–Ytm1 complex, the RNA helicase Has1 and Nop16) assemble with domain I surrounding ITS2 (Fig. 5a,d). These eight factors, together with several ribosomal proteins bound nearby, may help stabilize the very early folding achieved by base pairing of 5.8S rRNA with sequences in domain I (Supplementary Figure 2). Nine other assembly factors (Mak16, Rpf1, Nsa1, Rrp1, Ssf1 in complex with Rrp15, Rrp14 and Ebp2 in complex with Brx1) form a ring around the centre of particles in state 2/B to bridge domains I, II and VI to each other103 (Fig. 5a). Four other assembly factors (the GTPase Nog1, eIF6, Rlp24 and Mak11) bind to domain VI near the interface with domain V (Fig. 5a). Although most ribosomal proteins in rRNA domain I and domain II are already in their mature conformation in nucleolar particles, the assembly of these domains is finished only once the remaining assembly factors bound to them are released from pre- ribosomal particles. For example, assembly factors such as Ebp2–Brx1 are situated to prevent the premature formation of RNA– RNA interactions between domains I and V (Fig. 5a) and must be removed to enable downstream maturation steps. This transition nicely illustrates an important principle of ribosome assembly — the exit of assembly factors from pre- ribosomal particles is as important as their entry or their presence.

In the nucleolus, functional centres of the large subunit (the GAC, PTC and PET) begin to emerge during the transition from state 2/B particles to state C, D and E particles (Fig. 5a,b,d,e). For example, assembly factors Nop2, Nip7, Noc3 and the methyltransferase Spb1 bind to rRNA domains IV and V to aid in the formation of the PTC (Fig. 5b). Assembly of the rim around the exit of the PET is completed by release of assembly factors Ssf1–Rrp15 and Rrp14 and by stable association of ribosomal proteins L19, L25 and L31 (Fig. 4). Ssf1 is located in the same position in early nucleolar intermediates (Fig. 5a) as that of L31 in mature ribosomes. Thus, removal of Ssf1 along with Rrp15 and Rrp14 might allow the subsequent association of L31, L19 and L25 to complete the PET rim. However, it is not known what drives the release of these assembly factors to enable this molecular switch. During the transition from state D to state E, formation of the vestibule — the outer, wider portion of the PET — is enabled by exit of the assembly factor Rpf1. The amino- terminal portion of Rpf1 lies in what will become the vestibule, which is formed once Rpf1 departs. Rpf1 disassembles from early pre- rRNPs together with the adjacent proteins Mak16, Rrp1 and Nsa1 (Fig. 4). Most likely, release of these assembly factors is enabled by the direct removal of Nsa1 by the AAA- ATPase Rix7.

Considerable remodelling upon transit of pre-60S ribosomes from the nucleolus to the nucleoplasm. 
At least 9 assembly factors exit and 11 factors enter pre-60S particles during their transition from the nucleolus to the nucleus. This is evident in the differences between state E particles and Nog2 or Rix1–Mdn1 particles (Figs 4; 5b,c,e,f; 6c–e). These and the above- described protein exchanges trigger numerous downstream events, including further construction of functional centers such as the PTC and PET as well as the stable docking of the previously flexible central protuberance and cleavage of the ITS2 to initiate its removal. The PET undergoes additional steps of construction during the late nucleolar stage of large- subunit assembly, as Ebp2, Brx1, Noc3, Spb1, Nop2 and Nip7 exit and Nog2 and Rsa4 bind to domain V (Figs 5b,c; 6c,d). Comparison of the nucleolar state E particles and Nog2 reveals that in state E particles, assembly factors Spb1, Ebp2 and Noc3 block the formation of the mature structure of rRNA helices in domains II and V, which form a portion of the RNA walls of the mature PET. Thus, release of these factors during the transition from nucleolar particles in state E to Nog2 particles may enable the formation of a functional PET. The surroundings of Nog1 are also subjected to major remodelling during the transition from nucleolar pre-60S particles (Fig. 6c) through the late nucleolar Nog2 particle (Fig. 6d) to the nuclear Rix1–Mdn1-containing pre-60S particle (Fig. 6e). Especially noteworthy is the insertion of the carboxyterminal extension of Nog1 into the PET, almost back through to the PTC108. This observation may enable continued assembly of the tunnel, as during this interval, helices that form the walls of the tunnel transition from an immature unfolded state to the mature conformation. Alternatively, or in addition, the Nog1 extension might control proper assembly of the tunnel or prevent entry of other molecules. The central protuberance, atop the mature 60S subunit, includes the 5S RNP (ribosomal proteins L5 and L11 bound to 5S rRNA) and domain V helices 80 and  (refs3,4). Because 5S rRNA is transcribed from different genes from the other three rRNAs, it must separately assemble into pre-ribosomes. The dedicated chaperone Syo1 facilitates assembly of 5S rRNA with L5 and L11 in the cytoplasm and nucleoplasm, and the assembly factors Rpf2 and Rrs1 are necessary for association of this 5S rRNP with pre- ribosomal particles. Although the 5S RNP associates with the earliest pre-60S particles, presumably bound to central protuberance helices, it is not visible by cryo- EM until Nog2 particles are formed, most likely owing to the flexibility of the undocked central protuberance in the earlier assembly intermediates. Because the binding sites of Nip7 and Nop2 in early nucleolar particles are quite near or overlap those for Rpf2 and Rrs1 in later Nog2 particles104, the transition of the 5S rRNP to a more stable conformation in pre- ribosomes could depend in part on the displacement of the Nop2–Nip7 complex from pre-ribosomal particles before formation of Nog2 particles (Figs 5b,c; 6c,d). Rpf2, Rrs1, L5 and L11 are also required for assembly of Nog2 with domain V of rRNA113, suggesting that stable docking of the central protuberance may influence further construction of the PTC in domain V. Cleavage of the ITS2 is coupled with formation of the PET and the PTC and with docking of the central protuberance. The primary trigger for this cleavage seems to be the release of nearby assembly factors. The AAA- ATPase Mdn1 binds to the assembly factor Ytm1 and releases Ytm1 and the associated protein Erb1, the amino- terminal extension of which winds around pre-25S rRNA domain I proximally to ITS2. Assembly factors that interact with this long extension of Erb1 also exit at this time, and their exit presumably is coupled to the release of Erb1. Although we do not know how the endonuclease Las1 is specifically targeted to pre- ribosomal particles to cleave ITS2, mutations in ribosomal proteins or assembly factors (including Mdn1) that prevent release of these proteins, which are proximal to ITS2, also block its cleavage by Las1. This observation suggests that remodelling of rRNA domain I near ITS2 in pre-ribosomal particles is necessary to create a proper substrate for Las1. Compared with Las1, it is clearer how the exosome is recruited to pre-ribosomal particles to process ITS2 once it is cleaved. A mechanism that combines a molecular switch and molecular mimicry is employed, enabled by the remodelling of the domain around ITS2. Mtr4, which is the helicase component of the exosome, binds to the adaptor protein Nop53 in pre- ribosomal particles. Because both Erb1 and Nop53 use the same motifs to bind to assembly factor Nop7 in pre-ribosomal particles, release of Erb1 is necessary for binding of Nop53 and thus docking of the exosome. Interestingly, although cleavage of the C2 site in ITS2 is linked to earlier remodelling events, failure to cleave or process ITS2 does not appear to affect downstream steps of 60S subunit assembly until late cytoplasmic stages. Following depletion or inactivation of Las1 or Mtr4, the ITS2 is not removed. However, subunit maturation continues, although the resulting ribosomes retain the assembly factors Nop15, Cic1 and Rlp7 bound to ITS2. The ITS2-containing 60S subunits somehow bypass nuclear surveillance mechanisms and are exported to the cytoplasm but are defective in translation and are targeted for turnover by the ribosome quality control complex and the cytoplasmic exosome.


1. https://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S0167488909002651?via%3Dihub
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866757/#:~:text=In%20eukaryotic%20cells%20ribosomes%20are,and%20final%20rRNA%20processing%20steps.
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600399/
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958520/
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964341/
6. https://sci-hub.ren/10.1038/s41580-018-0078-y

Nucleoplasmic stages of large-subunit assembly. 
On reaching the nucleoplasm, pre-60S particles undergo additional remodelling steps as they become competent for export to the cytoplasm. These include removal of ITS2 and restructuring of the central protuberance as well as structural proofreading of functional centres. The ITS2 is removed sequentially by three nuclease complexes: the Las1–Grc3–Rat1–Rai1 complex, the exosome and Ngl2–Rex1-3. The endonuclease Las1 cleaves the C2 site in ITS2 to generate the 7S and 25.5S pre- rRNAs (Fig. 1). Trimming of the 25.5S pre- rRNA to 25S rRNA is enabled by the kinase Grc3 and is carried out by the nucleases Rat1 and Rai1. The 14-subunit exosome removes from the 3ʹ end of 7S pre- rRNA all but the most 5ʹ 30 nucleotides of the ITS2 and then in a second step all but the last 8 nucleotides to form the 6S pre- rRNA130. In the cytoplasm, Ngl2 and Rex1-3 catalyse processing of the 6S precursor to mature 5.8S rRNA. Formation of the mature central protuberance structure requires a large- scale structural rearrangement, which serves as a checkpoint in the nucleoplasm before nuclear export. The 5S RNP initially docks ‘backwards’ onto the top of pre-60S particles, then rotates ~180° to assume its mature configuration. At the same time, the accompanying central protuberance helices 80 and 82–88 also undergo a large- scale structural rearrangement. These remodelling events involve alterations of numerous protein–RNA contacts. Immediately before rotation of the central protuberance, Rpf2 and Rrs1 are released. These two assembly factors link the 5S RNP to the central protuberance helices in rRNA domain V at the top of the pre-60S ribosome. Thus, their exit might trigger destabilization of the prerotated state. Release of Rpf2 and Rrs1 may be induced by binding of the assembly factor Sda1 with preribosomal particles and by its competition with Rpf2 for partially overlapping binding sites (Fig. 6d,e). Following rotation, the Rix1–Ipi1–Ipi3 complex and the AAA- ATPase Mdn1 establish multiple contacts with the pre- ribosomal particle to stabilize the rotated state (Fig. 6e). ATP hydrolysis by Mdn1 activates the Nog2 GTPase, resulting in its release as well as the release of Sda1, Rix1–Ipi1–Ipi3, Mdn1 and Rsa4 (Fig. 4). To minimize production of dysfunctional ribosomes, yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly, which is coupled with export from the nucleus. The export factor Arx1 and its partner Alb1 assemble onto the rim around the PET by binding to ribosomal proteins L19, L25, L26 and L35. Thus, before export of pre- ribosomal particles, Arx1 binding may serve to proofread the proper accommodation of these ribosomal proteins into the tunnel exit, where a number of factors chaperone and target emerging nascent polypeptides during protein synthesis. The GTPase Nog2 and the adjacent GTPases Nog1 and Nug1 and RNA helicase Dbp10 may influence or scrutinize the proper formation of the PTC: following release of Nog2, this RNA neighbourhood looks much more like the mature PTC142. The departure of Nog2 enables assembly of the essential export factor Nmd3, the binding site of which stretches from the L1 stalk through the E site and P site towards the sarcin–ricin loop. Thus, the formation of an almost- mature PTC may signify export competence of pre-ribosomal particles. Maturation of the P0 stalk is also coupled with nuclear export: the assembly factor Yvh1 enables release of assembly factor Mrt4 from the premature stalk to enable binding of the export factors Mex67 and Mtr2.

SSU assembly: nucleocytoplasmic stages
The 3ʹ domain of 18S rRNA, which later forms the head of the mature small subunit, is subject to major remodeling steps between the nucleolar SSU processome and the cytoplasmic pre-40S particle (Fig. 6a,b). Whereas factors such as Enp1, Pno1 and Rrp12 remain associated with the 3ʹ domain, all other SSU processome factors are separated from the pre-40S subunit by a mechanism that is still poorly understood. Nuclear export of smallsubunit precursors occurs in a RanGTP- dependent and Crm1-dependent manner. In addition, nuclear pre-40S particles require Rrp12 for efficient export into the cytoplasm, and Rrp12 is absent from the cytoplasmic yeast intermediates that have recently been determined. During the transition from the nucleolus to the cytoplasm, pre-40S ribosomes also acquire a series of distinct nucleocytoplasmic assembly factors, including Tsr1, Rio2, Dim1 and Ltv1. Ltv1 replaces a peptide of Nop14 on the surface of Enp1, and the catalytically inactive Tsr1 replaces the structurally related GTPase Bms1 near the 5ʹ domain of the pre-40S particle (Fig. 2). Another important step is the formation of the beak structure of the small subunit, which in the mature structure is formed by rpS31, rpS12, rpS10, rpS3 and rpS20. In late pre-40S particles, Enp1 and the associated Ltv1 peptide block the access of rpS10; likewise, only the amino- terminal domain of rpS3 is incorporated into these structures. Pno1 not only binds to the 3ʹ end of the 20S pre- rRNA to assist in the cleavage at the D site by the flexibly attached Nob1 but also prevents the premature association of rpS26, which is a protein that is integrated with the help of its dedicated chaperone Tsr2. Very late steps of pre-40S assembly include testdriving the function of the subunit. Here, the dissociation of factors such as Tsr1 and Rio2 is thought to precede the association of Rio1 with the pre-40S particle. Together with a mature 60S subunit, eukaryotic translation initiation factor 5B and the ATPase Fap7 promote the formation of an 80S- like particle, which is used to functionally proofread the pre-40S particle, activate Nob1 for cleavage at the D site and remove late assembly factors such as Nob1 and Pno1 (Fig. 2).

Ribosome assembly and human diseases
In comparison with ribosome assembly in yeast, much less is known about how ribosomes are assembled in mammalian cells. A wide range of assembly factors involved in the synthesis of the human ribosome have been identified by large- scale screens, which have identified many homologues to proteins that were previously studied in yeast as well as new potential ribosome assembly factors. Defects in ribosome assembly in humans have been associated with cancer and with ribosomopathies, which comprise a heterogeneous set of diseases in which mutations of ribosomal proteins or assembly factors cause a variety of phenotypes. Ribosomopathies include Diamond–Blackfan anaemia (DBA), isolated congenital asplenia, North American Indian child cirrhosis, chromosome 5q– syndrome, Treacher Collins syndrome and Shwachman–Bodian–Diamond syndrome, among others. Despite the differences in their phenotypes, the biological underpinnings of these diseases can be subdivided broadly into two groups. The first is the triggering of a p53-dependent stress response and the second is the tissue- specific effect that has been observed in bone- marrow-derived cell lineages or skeletal tissues184. The activation of p53 due to defective ribosome assembly occurs through a complex containing the ribosomal 5S RNP. Importantly, the p53 response is a key cause of the phenotype of Treacher Collins syndrome, chromosome 5q– syndrome and North American Indian child cirrhosis. Mutations in ribosomal proteins cause their haploinsufficiency. In human cell lines, the depletion of ribosomal proteins can result in the accumulation of distinct ribosomal precursors. Although the largest number of mutations in DBA has been associated with ribosomal proteins, additional mutations were found in the rpS26 chaperone Tsr2. A more puzzling observation is the tissue- specific phenotype of DBA and other ribosomopathies. Early hypotheses included the presence of specialized ribosomes in these tissues, which would translate specific mRNAs; a more recent model proposed that limiting the number of ribosomes may affect particular sets of mRNAs, which are required for differentiation of particular tissues. Key pieces of evidence for this model include the identification of mutations in the haematopoiesis- specific transcription factor GATA1 in individuals with DBA and the demonstration that expression of GATA1 can at least partially compensate for haploinsufficiency of rpS19. More recent data show that mutations of ribosomal proteins reduce the pool of available ribosomes in DBA, which specifically affects the translation of a pool of transcripts that are necessary for erythroid lineage commitment. We believe that this may be a general mechanism, applicable to other ribosomopathies such as isolated congenital asplenia, in which the spleen does not form as a result of rpSA haploinsufficiency.


Eukaryotic ribosome assembly, transport and quality control  2017 1
Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes.


The ribosome translates the genetic code into proteins. In the eukaryotic model organism Saccharomyces cerevisiae, this universal two-subunit machine is composed of a large 60S subunit, consisting of three rRNAs (25S, 5.8S, 5S) and 46 different ribosomal proteins (r proteins), and a small 40S subunit, consisting of 18S rRNA and 33 r proteins. In contrast to that of their bacterial and archaeal counterparts, assembly of eukaryotic ribosomes requires more than 200 conserved assembly factors, including diverse RNA-binding proteins, endo- and exonucleases, RNA helicases, GTPases and ATPases. These assembly factors promote pre-rRNA folding and processing, remodeling of protein–protein and RNA–protein networks, nuclear export and quality control.

Eukaryotic ribosome assembly is a complex process spanning various cellular compartments (Box 1). It starts with transcription of a single RNA polymerase (Pol) I transcript, the 35S pre-rRNA, in the nucleolus. This transcript undergoes several processing steps to eventually give rise to mature 18S, 5.8S and 25S rRNAs (Fig. 1).


Figure 1 Pre-rRNA processing pathway. 
The Pol I–transcribed 35S prerRNA contains sequences for 18S, 5.8S and 25S rRNA, flanked and separated by external transcribed spacers (ETS) and internal transcribed spacers (ITS) (indicated in red). Pre-5S rRNA is transcribed by RNA Pol III. A series of co- and post-transcriptional endo- and exonucleolytic events removes the spacer sequences to yield mature rRNA (prerRNA intermediates are indicated in blue, enzymes and corresponding processing sites in green).

 In the 35S pre-rRNA, 18S rRNA is preceded by a 5′ external transcribed spacer sequence (5′ ETS) and followed by the internal transcribed spacer sequence ITS1 which, together with ITS2, separates 5.8S rRNA from the downstream 25S rRNA. Cotranscriptional cleavage at the A1 and A2 sites releases the 20S pre-rRNA, associated assembly factors and r proteins as the earliest 40S preribosome. The remaining 27S A2 pre-rRNA recruits 60S-specific r proteins and assembly factors to form the earliest pre-60S ribosome. 60S pre-ribosomes undergo sequential pre-rRNA processing to yield mature 25S and 6S rRNA before nuclear export. Exported 40S pre-ribosomes containing 20S pre-rRNA undergo final RNA processing to yield mature 18S rRNA, and 6S pre-rRNA within exported 60S pre-ribosomes gets trimmed to mature 5.8S rRNA. A critical task for the ribosome assembly machinery is to precisely incorporate r proteins onto dynamically folding pre-rRNA. This is a logistical challenge given that r proteins, which are synthesized in the cytoplasm, need to be targeted to the nucleus and then handed over to the ribosome assembly machinery. Recent studies have provided mechanistic insights into how r proteins are guided to their rRNA-binding site on the maturing preribosome. Moreover, advances in cryo-EM revealed structural snapshots of different stages of eukaryotic ribosome assembly, enabling further hypothesis-driven functional studies.

Targeting r proteins to assembling preribosomes
During a single 90-min generation, a yeast nucleus imports ~14 million r proteins for assembly and at the same time exports 200,000 preribosomes to the cytoplasm. Rapid transport of r proteins, assembly factors, and preribosomes through nuclear pore complexes (NPCs) is essential for ribosome production. In yeast, Kap104, Pse1 and Kap123 are the primary importins that target r proteins to the nuclear compartment. Unlike typical import cargos, r proteins are disordered, which makes them prone to nonspecific interactions with nucleic acids, aggregation and degradation in their nonassembled state. Thus, nuclear import of r proteins and their subsequent transfer to their rRNA-binding site on the assembling preribosome poses a challenge. Another challenge for the ribosome assembly machinery is to ensure that stoichiometric levels of r proteins are incorporated into every preribosome before nuclear export. The ribosome-anchored NAC and SSB–RAC chaperones stabilize aggregation-prone r proteins in the cytoplasm before their nuclear import. Simultaneous inactivation of NAC and SSB–RAC induces aggregation of r proteins of both ribosomal subunits. Apart from these chaperone systems, importins, in addition to their role in mediating their nuclear import, also stabilize r proteins. Owing to the high demand for r proteins during ribosome formation, however, these factors are not sufficient, and specialized mechanisms  protect r proteins during their journey to the nucleolus. This is achieved by a functional class of dedicated chaperones that interact with newly synthesized r proteins and facilitate their nuclear import and/or escort them to preribosomes. At least nine r proteins, uS3 (Rps3), uS11 (Rps14), eS26 (Rps26), uL3 (Rpl3), uL4 (Rpl4), uL14 (Rpl23), uL16 (Rpl10), uL18 (Rpl5) and uL5 (Rpl11), are bound by dedicated chaperones in vivo. Affinity purification of several of these chaperones selectively enriches the mRNA encoding their r-protein client, suggesting that r proteins are captured as they emerge from the translating ribosome12. However, it is still unclear how dedicated chaperones identify their specific target r proteins at translating ribosomes. Below, we review several of these dedicated chaperones and the mechanisms by which they target r proteins to assembling ribosomes (Fig. 2).


Figure 2 Targeting r proteins to assembling preribosomes. 
(a) Yar1 captures the N terminus of uS3 (Rps3), which dimerizes with a second uS3 molecule to allow importin recruitment. After nuclear import, the uS3–uS3–Yar1 complex is dissociated to incorporate uS3 into the pre-40S subunit. 
(b) Rrb1 cotranslationally captures uL3 (Rpl3) and accompanies the r protein into the nucleus to its assembly site on the pre-60S. 
(c) Sqt1 binds the nascent N terminus of uL16 (Rpl10) in a cotranslational manner and assists uL16 incorporation into pre-60S subunits in the cytoplasm. 
(d) Syo1 captures uL18 (Rpl5) cotranslationally and binds uL5 (Rpl11) post-translationally. Kap104 then binds to Syo1 to mediate synchronized nuclear import of the r proteins. After recruitment of the 5S rRNA to the Syo1–uL18–uL5 complex in the nucleus, Syo1 is released and the 5S RNP is incorporated into pre-60S subunits. 
(e) Acl4 cotranslationally binds to a long internal loop in uL4 (Rpl4). A second copy of Acl4 recognizes the C terminus of uL4 before being displaced by Kap104 to mediate nuclear import of Acl4–uL4. In the nucleus, Kap104 dissociates in a Ran-dependent manner to allow rebinding of the second Acl4 copy. Acl4 dissociates upon incorporation of uL4 into pre-60S subunits. 
(f) eS26 (Rps26) is imported into the nucleus and then picked up from its importin in a Ran-independent manner by the escortin Tsr2. Fap7 and uS11 (Rps14) then join eS26 to prefabricate a ribosomal subcomplex that allows stoichiometric incorporation of uS11 and eS26 into the 90S. 
(g) uL14 (Rpl23), bound to its importin, is imported into the nucleus and then released in a Ran-dependent manner. Bcp1 then picks up uL14 to escort the r protein to its assembly site on the pre-60S.

Yar1, an ankyrin-repeat protein, binds to the N-terminal domain of uS3 in the cytoplasm and accompanies the r protein into the nucleus (Fig. 2a). Surprisingly, uS3 contains a nuclear localization signal (NLS) in its N domain that interferes with the Yar1-binding site and recruits the classical importin dimer Kap60–Kap95. However, when associated with Yar1, the C-terminal domain of uS3 interacts with a second copy of uS3. This allows binding of Yar1 to one N-terminal domain and Kap60 to the second N-terminal domain of dimerized uS3, thereby orchestrating simultaneous nuclear import and protection of this r protein. Rrb1 and Sqt1 are both WD-repeat β-propeller proteins that bind to uL3 and uL16, respectively (Fig. 2b,c). Rrb1 localizes to the nucleolus, and its overexpression leads to nuclear accumulation of uL3. Translation inhibition mislocalizes Rrb1 to the cytoplasm. This suggests that Rrb1 accompanies uL3 into the nucleus, but it remains unclear how their nuclear import is mediated. In contrast, uL16 is incorporated into the late cytoplasmic pre-60S subunit, and therefore the Sqt1–uL16 pair is not imported into the nucleus. Structural analysis of Sqt1 bound to the N-terminal extension of uL16 suggests that Sqt1 protects the N terminus of uL16 during its incorporation into the preribosome. Release of Sqt1 might then allow the N-terminal extension of uL16 to stably incorporate into its cognate binding site helix H89 of the 25S rRNA. Syo1 adopts an elongated α-solenoid fold through an unusual combination of four armadillo repeats and six HEAT repeats. It is unique in that it functions as import adaptor for two different r proteins, uL18 (Rpl5) and uL5 (Rpl11), via two different binding sites (Fig. 2d). An N-terminal proline-tyrosine-NLS in Syo1 recruits the importin Kap104 and facilitates simultaneous coimport of the r-protein clients. Upon arrival in the nucleus, the trimeric Syo1–uL18–uL5 cargo complex is released in a RanGTP-dependent manner. Whether coimport of uL18 or uL5 into the nucleus is affected in Syo1 mutants that are selectively impaired in binding these r proteins, however, remains unclear. uL18 and uL5 are functionally related and, together with 5S rRNA, they are incorporated into early pre-60S subunits as part of the 5S ribonucleoprotein (RNP) complex. The Syo1–uL18–uL5 trimer therefore serves as an assembly platform to allow 5S rRNA recruitment to form a pre-5S RNP. In this complex, Syo1 binds to the same surface on uL5 that interacts with helix H84 of 25S rRNA, suggesting that Syo1 protects this binding site before being replaced by helix H84. It is unclear, however, if Syo1 remains associated with the 5S RNP during its incorporation into pre-60S subunits. Acl4 is a tetratricopeptide-repeat-containing protein that, unlike the aforementioned dedicated chaperones, does not bind to the N-terminal region of uL4 (Fig. 2e). Instead, one copy of Acl4 binds to a long internal loop in uL4, while a second copy of Acl4 binds to its eukaryotic-specific C-terminal extension. Reminiscent of Yar1–uS3 nuclear import, the C-terminally bound copy of Acl4 can be displaced by the importin Kap104 to form an import-competent heterotrimeric complex. Upon arrival in the nucleus, Acl4–uL4 is released from Kap104 in a Ran-dependent manner, thereby permitting its incorporation into the 60S preribosome. Thus, like Yar1, Acl4 has a dual function to facilitate nuclear import and protect unassembled uL4. How the release of an r protein from its dedicated chaperone is coupled to its incorporation into the assembling preribosome remains unknown. In case of Acl4–uL4, an interaction of the uL4 eukaryotic- specific C-terminal extension with eL18 on the 60S preribosomal surface is thought to trigger energy-independent disassembly of the Acl4–uL4 complex and allow uL4 incorporation21. In contrast, incorporation of uS11 is catalyzed by the essential chaperone Fap7 in an ATP-dependent manner. Fap7 binds to and stabilizes uS11 both in vivo and in vitro24–27. The ATPase activity triggers uS11 release onto helix H23 within 18S rRNA26,27. Structural analysis of the Fap7–uS11 complex revealed that Fap7 serves as an RNA mimic for helix H23, the binding site of uS11 in mature 40S subunits25,26. RNA mimicry may provide a surrogate platform to fold an r protein and prepare it for loading on the preribosome. In addition, complex formation with a dedicated chaperone may prevent nonproductive interactions between the r protein and other cellular RNAs.

In contrast to r proteins described above, a different mechanism is employed by the r protein eS26 to reach the 90S preribosome (Fig. 2f). eS26, like a typical import cargo, directly binds to importins for targeting to the nucleus. However, unlike a typical import cargo, eS26 is extracted in the nucleus from the importin by an unloading factor, the escortin Tsr2, without the aid of RanGTP. Once bound to Tsr2, eS26 is shielded from proteolysis, enabling its safe transfer to the 90S preribosome. Intriguingly, in mature ribosomes, eS26 is located between the r protein uS11 and the very 3 end of 18S rRNA2. Although uS11 could be readily assigned in recent cryo-EM structures of 90S pre-ribosomes, eS26 could not be visualized in these structures, even though mass spectrometric analysis indicates its presence on the yeast 90S. It is conceivable that eS26 has not achieved its native state, thus precluding the reliable modeling of its native structure on the 90S. Alternatively, missing densities at the very 3′ end of 18S rRNA, following ITS1 on the 90S, suggest that eS26 might be present in a flexible region, making it refractory to structural studies. Recently, Bcp1 was shown to function as an escortin for the r protein uL14 (Rpl23) by extracting uL14 from its importins Kap121 and Kap123 in a RanGTP-dependent manner32 (Fig. 2g), but how eS26 and uL14 are released from their escortins remains unclear. How does the ribosome assembly machinery guarantee stoichiometric integration of r proteins into pre-ribosomes? At least two different mechanisms seem to ensure that correct levels of r proteins are delivered to the preribosome. The symportin Syo1 has been proposed to coordinate the transfer of spatially proximal r proteins uL18 and uL5 onto 5S rRNA (Fig. 2d). Here, Syo1 employs separate binding sites to directly bind r proteins uL18 and uL5 and synchronize their import for 5S RNP assembly. The second mechanism involves the prefabrication of ribosomal protein complexes, as exemplified by the ATPase Fap7 that assembles a native-like uS11–eS26 ribosomal protein complex before its stable incorporation into the 90S preribosome (Fig. 2f). uS11, when bound to Fap7, directly recruits eS26 through tertiary contacts found between these r proteins on the mature 40S subunit. Mutations in these conserved contacts preclude stoichiometric prefabrication of the uS11–eS26 complex. Fap7 ATPase activity then unloads the uS11–eS26 complex onto rRNA helix H23. In addition, several r proteins cluster on the surface of the ribosome through a network of intricate tertiary contacts. Prefabricating these r-protein subcomplexes through tertiary contacts before their assembly also ensures accuracy of ribosome production. In the absence of dedicated chaperones, orphaned r proteins are efficiently recognized and degraded by the cellular protein quality-control machinery. Recent work has implicated the HECT-domain E3 ubiquitin ligase Tom1 in clearing excess r proteins. Interestingly, Tom1 ubiquitinates unassembled r proteins through residues that are inaccessible in mature ribosomes and targets them for proteasome-dependent degradation. This degradation pathway, termed ERISQ, for excess ribosomal protein quality control, may be one mechanism employed to maintain cellular homeostasis of r proteins.

Small subunit assembly and acquisition of export competence
Early nucleolar preribosome assembly occurs dichotomously: production of the pre-40S subunit is followed by pre-60S subunit synthesis. Initially, a large RNP complex, referred to as 90S or the small subunit (SSU) processome, assembles on the emerging RNA Pol I–transcribed 5′ ETS and 18S rRNA to form the characteristic terminal knobs seen on Miller spreads. The 90S assembly process is aided by ~70 assembly factors and small nucleolar RNAs (snoRNAs), most prominently the U3 snoRNP. The 90S complex consists of structurally autonomous subcomplexes and proteins that join the nascent 5′ ETS and 18S and ITS1 rRNA in a sequential manner.
Recent work has elucidated in more detail the temporal events leading to the pre-40S subunit formation. Further, three independent cryo-EM studies have provided insights into the earliest steps of pre-40S subunit assembly (Fig. 3).


90S pre-ribosome assembly. 
(a) Cryo-EM structure of the S. cerevisiae 90S preribosome. 18S rRNA is colored in gray; all yellow regions depict r proteins (model shows a composite structure based on PDB 5TZS and PDB 5WYK). 
(b) The 90S complex consists of structurally autonomous subcomplexes and proteins that join the nascent 5′ ETS and following 18S and ITS1 rRNA in a sequential manner. The order of events (top) is represented by highlighting the subcomplexes in distinct colors based on their association with the emerging pre-rRNA (depicted at the bottom). min, minor.

First, the UtpA subcomplex interacts with several RNA helices formed by the 5′ ETS and primes it to recruit U3 snoRNP and the UtpB modules. UtpA and UtpB are related multiprotein subcomplexes that scaffold the SSU processome to subsequently assemble the 18S rRNA-containing pre-40S ribosome. UtpA is composed of seven subunits, of which two contain tandem β-propellers (Utp4 and Utp17) and four contain one β-propeller and an α-helical C-terminal domain (Utp5, Utp8, Utp9 and Utp15), which induces oligomerization. Utp10, the last subunit, is a large helical repeat protein that wraps around the UTP modules. In contrast, UtpB is composed of six subunits, with Utp1, Utp12, Utp13 and Utp21 forming a tetramer of tandem β-propeller proteins containing an α-helical C-terminal domain. The remaining subunits, Utp6 and Utp18, are located in proximity to the U3 snoRNP, which undergoes extensive base-pairing interactions with regions of the 5′ ETS as well as the beginning of 18S rRNA. Together with the Mpp10–Imp3–Imp4 complex and additional large helical proteins, these 5′ ETS components create an assembly platform that hugs the emerging 18S rRNA domains and spatially segregates them to facilitate access of enzymes and assembly factors to the 90S core. Chaperoned by the UtpC complex, the emerging 5′ domain of 18S rRNA allows recruitment of a first set of 40S r proteins. Subsequently, the core body of the 90S is formed by recruiting a subset of assembly factors including the GTPase Bms1, the methyltransferase Emg1, the homodimeric acetyltransferase Kre33 and the large 288-kDa α-helical Utp20. Bms1 forms a complex with Rcl1 and has been proposed to catalyze cleavage at the A2 site. However, within the 90S, Bms1–Rcl1 bridges distant 5′ and 3′ domains of 18S rRNA, which does not support a direct role in cleavage. Emg1 methylates 18S rRNA at nucleotide position 1,191, and Kre33 acetylates 18S rRNA nucleotides 1,280 and 1,773. Whereas Emg1 is readily positioned toward nucleotide 1,191, Kre33 is located far away from its substrates. It is therefore possible that the activities of Bms1–Rcl1 and Kre33 might require global pre-rRNA rearrangements. Utp20 and additional unresolved helical-repeat proteins may act as mediators of long-range interactions to stabilize the 90S. In its final form, the SSU processome contains the PIN-domain nuclease Utp24 that has been suggested to catalyze cleavage at the A0 and A1 site and release the 5′ ETS particle from the assembled pre-40S. In yeast, cleavage at site A2 predominantly occurs cotranscriptionally and releases the early pre-40S particle containing 20S pre-rRNA into its independent nuclear assembly pathway51. Under unfavorable growth conditions, however, the pre-rRNA is cleaved at site A3 to generate 23S pre-rRNA. This process is regulated by the TOR1 pathway. How exactly the 90S transitions into the pre-40S stage remains unclear from a structural viewpoint. A comparison between the 90S-associated 18S rRNA and its mature fold suggests that only the 5′ domain of 18S rRNA has adopted its final conformation. It is possible that the central and 3′ domains fold into their mature configuration after release of the 5′-ETS particle. In general, the structural landmarks as seen in the mature 40S subunit have yet to achieve a compacted state in the 90S subunit. It appears that the 90S has a perforated structure with numerous cavities and channels in its inner core. Nuclear pre-40S particles acquire export competence through the recruitment of a distinct set of assembly factors, Enp1, Dim1 and Pno1, which join during the 90S stage, and Tsr1, Rio2, Ltv1, Hrr25 and Nob1, probably joining after A2 cleavage. Prior to nuclear export, pre-40S subunits undergo an essential maturation step during which the binding of r protein uS3 (Rps3) to the ‘beak’, a structural landmark in the head domain of mature 40S subunits, is reorganized. In these pre-40S particles, a subcomplex composed of the assembly factors Enp1, Ltv1 and uS3 is bound in proximity to the beak structure55. Phosphorylation of Enp1 and uS3 by the kinase Hrr25 weakens their affinity for the pre-40S and increases the conformational flexibility of the head. It has been proposed that beak flexibility might be critical for nuclear pre-40S particles to acquire export competence. One of the earliest cryo-EM studies on ribosome assembly allowed mapping of Enp1, Ltv1, Tsr1, Rio2, Dim1, Pno1 and Nob1 on a late 40S pre-ribosome.

Large subunit assembly and acquisition of export competence
Release of the pre-40S particle permits the remaining pre-rRNA to assemble into a pre-60S subunit. Early assembly of the pre-60S follows principles similar to those of the pre-40S. Using plasmid-encoded pre-rRNA fragments of increasing lengths, a stepwise association of assembly factors and ribosomal proteins with emerging 27S pre-rRNA of the pre-60S subunit was demonstrated. This supports the idea that, like the 90S, the early pre-60S is assembled cotranscriptionally rather than post-transcriptionally.  A myriad of assembly factors are implicated in nuclear pre-60S maturation, including snoRNPs, RNA helicases and RNA-modifying or RNA-processing enzymes. Together, they are involved in further cleavage and trimming of 27S rRNA to remove most of the ITS1, ITS2 and 3′-ETS sequences. In addition, the remaining 5S RNP is recruited, and the pre-60S is compacted into its characteristic structure. In contrast to early pre-60S assembly, subsequent steps leading to acquisition of export competence are amongst the best characterized. One of the first cryo-EM reconstructions representing a nuclear pre-60S intermediate, the ‘Arx1 particle’, revealed regions of extra densities attributed to associated biogenesis factors. Named after its association with the assembly factor Arx1, this pre-60S particle exhibits characteristic structural landmarks, such as a large ‘foot’ structure originating from the unprocessed ITS2-containing 7S pre-rRNA and, importantly, a 5S RNP and a central protuberance that are rotated ~180° compared to a mature 60S particle. The Woolford and Gao labs were able to assign densities to several assembly factors on a nucleoplasmic particle isolated using the conserved GTPase Nog2 (Nug2) as bait62 (Fig. 4a).



Figure 4 Nuclear pre-60S assembly. 
Cryo-EM structures of (a) Nog2 and (b) Rix1 particles representing two late nuclear pre-60S intermediates. The Nog2 particle represents an earlier assembly intermediate in which the ITS2 spacer has not yet been removed and the 5S rRNA is still in a premature conformation. In the Rix1 particle, ITS2 is removed and the 5S rRNA has rotated into its mature position upon recruitment of the Rix1 subcomplex and Rea1. 25S rRNA is depicted in gray, 5.8S and 5S in black, ITS2 in brown and r proteins in yellow. (Nog2 PDB 3JCT, ref. 62; Rix1 PDB 5Fl8, EMDB-3199, ref. 64.) 
(c) Scheme for acquisition of export competence for the large pre-60S subunit. Only assembly factors with known binding sites are indicated.

The structure of this particle, which partially overlaps with the Arx1 particle, revealed assembly factors involved in building the ITS2-containing foot structure. The foot structure is established through an intricate interaction network involving the assembly factors Nop15, Cic1, Rlp7, Nop7 and Nop53. Processing of 7S pre-rRNA, and thus removal of the foot structure, is carried out by the exosome and requires prior release of Nop15, Cic1 (also known as Nsa3) and Rlp7. Nop53 initiates processing of the 7S pre-rRNA by recruiting the exosome-associated helicase Mtr4; however, how these events are coordinated remains unclear.

The structure of the Nog2 particle also revealed how the rotated 5S RNP is docked on the assembly factors Rpf2, Rrs1 and Rsa4. To acquire export competence, the 5S RNP has to rotate ~180° into its final position. This dramatic remodeling step is likely carried out through recruitment of the Rix1 subcomplex and its interactor Rea1, a dynein-related 550-kDa AAA-ATPase. A cryo-EM study using Rix1 as bait revealed the structural landscape of how these two factors form a checkpoint for acquisition of export competence (Fig. 4b). In the Rix1 particle, the 5S RNP is rotated into its mature position where it contacts Rix1. Thus, recruitment of Rix1 is thought to destabilize the immature 5S RNP conformation, thereby inducing its rotation. Only after accurate 5S RNP rotation can the huge Rix1–Rea1 machinery be stably anchored through multiple contact points on the pre-60S. Correct positioning of Rea1 has been suggested to trigger its ATPase activity, which motors the removal of Rsa4 by an ATP-hydrolysis-driven “power stroke”. Intriguingly, the Rea1 ATPase activity is also coupled to the release of Nog2, a K+-dependent GTPase whose enzymatic activity is required for its own release. Nog2 is a placeholder for Nmd3, an essential assembly factor required for nuclear export of pre-60S subunits. Thus, the Rix1–Rea1 machinery and the coupled enzymatic activities of Rea1 and Nog2 may serve as a checkpoint to render pre-60S subunits competent for nuclear export (Fig. 4c).

Nuclear export of pre-ribosomes
In growing yeast cells, every minute, ~25 preribosomal particles are exported into the cytoplasm, with the exportin Crm1 (yeast Xpo1) playing an essential role the transport. To initiate export, Crm1, in the presence of RanGTP, needs to recognize a nuclear export signal (NES) on adaptor proteins bound to preribosomes and cooperatively form a Crm1-export complex. Nmd3 is an essential export adaptor protein for Crm1-dependent export of the 60S preribosome. It contains a bipartite leucine-rich NES in its C-terminal domain that is essential for yeast viability. Overexpression of nmd3ΔNES leads to dominant-negative effects and impairs pre-60S-subunit export. Two recent cryo-EM structures of pre-60S subunits have revealed how Nmd3 is bound to a ribosome69,70. Purification of native Nmd3-TAP from yeast led to a structural snapshot of a cytoplasmic pre-60S containing Nmd3 and the assembly factors Lsg1 (Kre35), Tif6 and Reh1 (ref. 69). Using a different approach, Malyutin et al. reconstituted mature 60S with recombinant Nmd3, Lsg1 and Tif6 and were able to solve most of the Nmd3 structure. Interestingly, the NES-containing C terminus of Nmd3 was not resolved in either study, supporting the idea that recruitment of Crm1 occurs via a flexible region. In contrast to Nmd3-dependent pre-60S export, no essential NES-containing export adaptor for pre-40S export has been identified. However, the assembly factors Ltv1 and the atypical kinase Rio2 carry a leucine-rich NES at their C termini that recruits Crm1 in the presence of RanGTP, suggesting that there are redundant mechanisms for 40S preribosome export. A common feature of NESs is their low affinity toward Crm1, even in the presence of RanGTP. Given the rate of preribosome export, eukaryotes must have mechanisms to efficiently assemble Crm1-export complexes to boost preribosome export. For pre-60S export, Nmd3 may employ a bipartite NES and enhance recruitment of Crm1. It is plausible that a Crm1 dimer is loaded to the NESs, as has been reported for the Rev oligomer that functions in HIV RNA export73. Pre-40S subunits appear to employ two RanGTP-binding proteins, Slx9 and Yrb2, to promote assembly of the Crm1-export complex. Slx9 was identified as a RanGTP-binding protein that facilitates recruitment of Crm1 to Rio2 (ref. 74). In vitro, Slx9 binds Rio2 and RanGTP to form a ternary complex, which then directly recruits Crm1. Slx9 seems to prime the Rio2 NES and possibly orient RanGTP in such a manner as to enhance Crm1 loading and boost pre-40S export. Yrb2 is another RanGTP-binding protein that enhances assembly of Crm1-export complexes. Yrb2 uses its two hydrophobic FG-repeat domains to bind Crm1 and its C terminus to bind RanGTP, thus forming a Yrb2–RanGTP–Crm1 complex. In this ternary complex, the NES-binding cleft of Crm1 is in a closed conformation. Loading of the NES onto the NES-binding cleft readily displaces the C terminus of Yrb2 from the complex through an allosteric mechanism. FRET analyses of this Yrb2–RanGTP–Crm1–NES complex further showed that binding of the NES to Crm1 is accelerated in the presence of Yrb2. Unlike Slx9, Yrb2 primes RanGTP and Crm1 for rapid loading onto NES-containing cargos. Curiously, yrb2Δ cells are impaired in pre-40S-subunit export but not pre-60S export. However, the precise targets of Yrb2 to boost 40S preribosome export remain unknown.

In addition to Crm1-dependent export adaptors, preribosomes recruit auxiliary factors that directly interact with the NPC. The Mex67–Mtr2 heterodimer and Rrp12 have been identified as common factors that promote the nuclear export of pre-60S and pre-40S particles. UV crosslinking and analysis of cDNA demonstrated that Mex67 interacts in vivo with the 20S pre-rRNA and 5.8S rRNAs. Reconstitution of a late 60S preribosome with Mex67–Mtr2 in vitro and identification of additional crosslinks to H42-43 of the 25S rRNA79 suggests two separate binding sites for this transport receptor on the 60S preribosome. Additional factors Arx1, Ecm1, Bud20 and Gle2 also facilitate efficient export of the 60S preribosome. Although these export factors are nonessential, their deletions are synthetically lethal when combined with each other or additional export mutants. These genetic interactions suggest redundant roles of export factors and support the idea that the large preribosomal cargos require multiple cooperative interactions for translocation through the NPC channel3.

Cytoplasmic quality control of the ribosome
Upon arrival in the cytoplasm, preribosomal particles undergo late maturation before achieving translation competence. These final steps involve further processing of pre-RNA, incorporation of remaining r proteins and release of associated assembly and transport factors. Cytoplasmic maturation also provides a time window for the quality-control machinery to functionally proofread preribosomes. In the case of pre-60S subunits, energy-consuming enzymes trigger these steps in a stepwise fashion. The AAA-ATPase Drg1 initiates cytoplasmic maturation of the 60S preribosome by specifically binding and releasing the ribosomal-like protein Rlp24, a placeholder for the r protein eL24. Expression of a dominant-negative allele of Drg1 traps cytoplasmic pre-60S subunits by blocking the recycling of not only Rlp24 but further assembly factors including Nog1, Mrt4, Tif6, Bud20, Nmd3, Mex67 and Nsa2. This Drg1-dependent event is therefore critical for ribosomal tunnel maturation and stalk assembly. The presence of r protein eL24 on the 60S preribosome triggers Rei1 and Jjj1 recruitment to the ribosomal exit tunnel, which is sealed by Arx1. Jjj1 is a J-domain protein that binds in the immediate vicinity of the Arx1–Rei1 interaction site and recruits and activates the Hsp70-type ATPase Ssa1 or its paralog Ssa2, inducing Arx1 release. Structural analyses of a reconstituted 60S subunit with Arx1, Rei1 and Jjj1 revealed how Rei1 probes the ribosome polypeptide tunnel (Fig. 5).


Figure 5 Cytoplasmic quality control of the pre-60S subunit. 
Cryo-EM structures of S. cerevisiae 60S subunits reconstituted with 
(a) Rei1, Arx1, Alb1 (PDB 5APN), 
(b) Nmd3, Lsg1, Tif6 and 
(c) D. discoideum 60S subunits reconstituted with human EFL1, human Sdo1 (SBDS) and endogenous eIF6 (Tif6). 
(d) Scheme for cytoplasmic quality control of the pre-60S subunit. Only assembly factors resolved in a–c are shown. The C terminus of Rei1 is inserted into the polypeptide exit tunnel (PET). PTC, peptidyl transferase center; CP, central protuberance.

Release of Nmd3 seems to be tightly coupled with release of Tif6, an assembly factor that prevents pre-60S ribosomes from interacting with mature 40S subunits. It has been proposed that recruitment of the assembly factors Efl1 and Sdo1 disengages Nmd3 from Tif6. This allows Efl1 and Sdo1 to promote the release of Tif6 from pre-60S ribosomes. A cryo-EM study in which human Sdo1 (SBDS) and Efl1 were bound to Dictyostelium discoideum 60S subunits revealed that upon binding of Efl1, Sdo1 is repositioned around helix H69, thus facilitating a conformational switch in Efl1 that displaces Tif6 (Fig. 5). Efl1 shares sequence similarity with the GTPase elongation factor Ef-2. Thus, Efl1 could check the integrity of the GTPase-activating center, while Sdo1 proofreads the PTC of the ribosome. In this way, cytoplasmic release factors may couple the recycling of shuttling assembly factors and simultaneously check ribosome function. Similar to the pre-60S export factors, recycling of pre-40S export factors Ltv1 and Rio2 is energy dependent. Ltv1 is released through phosphorylation by Hrr25, the same kinase that is also involved in conferring export competence for the pre-40S in the nucleus. In contrast, Rio2 uses its intrinsic ATPase activity to dissociate from the pre-40S94. Release of Rio2 allows recruitment of Rio1, a related ATPase implicated in final quality control of the 40S subunit. Cryo-EM studies of a late 40S preribosome assigned binding sites for multiple assembly factors including Enp1, Ltv1, Nob1, Pno1, Dim1, Rio2 and Tsr1 (Fig. 6). 


Figure 6 Cytoplasmic quality control of the pre-40S subunit. 
(a) Cryo-EM structure of the Rio2 particle representing a cytoplasmic pre-40S ribosome. Assembly factors and 18S rRNA helix 44 are depicted in colors, and remaining rRNA and r proteins are shown in gray.
(b) Scheme for cytoplasmic quality control of the pre-40S subunit. Only assembly factors with known binding sites are indicated.

These analyses suggest that these assembly factors prevent premature translation initiation by inhibiting access of translation factors. Ltv1 and Enp1 directly bind uS3 (Rps3) on its solvent-exposed side, thereby blocking the opening of the mRNA channel. Nob1 and Pno1 inhibit the binding of eIF3 and thereby interfere with translation initiation. Dim1, Rio2 and Tsr1 are localized at the subunit interface, thus preventing premature interactions with the 60S subunit and translation initiation factor eIF1A. Moreover, Tsr1 acts as an inactive structural mimic of eIF5B (Fun12), a GTPase involved in the final steps of pre-40S maturation. The location of Tsr1 is further predicted to interfere with binding of Rio1, strongly indicating that removal of both Rio2 and Tsr1 is essential for 40S maturation.Release of Rio2 and Tsr1 then allows the recruitment of Rio1 and Fun12. The resulting pre-40S intermediate appears to mimic the translation-initiation surface of a 40S subunit, because it allows the interaction with a mature 60S subunit to form an 80S-like particle (Fig. 6). Curiously, formation of the 80S-like particle containing active Rio1 and Fun12 stimulates release of Pno1 and triggers the endonuclease activity of Nob1, suggesting that 20S pre-rRNA processing may be a quality-control mechanism triggered only on translation-competent 40S pre-ribosomes.

Eukaryotic Ribosome Assembly and Nuclear Export 04 Aug 2015 2
Synthesis of ribosomes is a central cellular event that consumes significant amounts of metabolites and energy. Two polymerases generate rRNAs in parallel: RNA Polymerase I and RNA Polymerase III. In addition,
RNA Polymerase II is required to transcribe the 139 ribosomal protein genes (RPGs) in a tightly regulated manner. Notably, 102 of the 139 RPGs in budding yeast contain introns. Although such intron-containing genes represent less than 5% of genes in yeast, they account for nearly one-third of total cellular transcription, making splicing a crucial cotranscriptional process. In total, the concerted activity of all three-transcriptional machineries (RNA polymerases I, II, and III), the splicing apparatus and the cellular transport system are required to ensure highly efficient and accurate ribosome biogenesis. In addition to rRNA and r-protein components, eukaryotic ribosomal subunit assembly requires >350 nonribosomal factors

Assembly and nuclear export of pre-ribosomal particles in budding yeast   11 May 2014 3
Correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. 

Error-free protein synthesis is vital for optimal cellular growth and proliferation, a fundamental task carried out by the ribosome. The small subunit decodes the genetic information by bringing together the messenger RNA (mRNA) template and cognate transfer RNAs (tRNAs). The large subunit catalyzes the peptidyl transfer reaction to synthesize the nascent polypeptide chain. Despite a similar core, eukaryotic ribosomes are significantly larger than their prokaryotic counterparts. The large ribosomal subunit (60S) in yeast contains three rRNAs (25S, 5.8S, 5S) and 46 r-proteins, whereas the small subunit (40S) contains one single rRNA (18S) and 33 rproteins. The assembly of the eukaryotic ribosome is a highly dynamic process that occurs in different cellular compartments: the nucleolus, the nucleoplasm, and the cytoplasm. In contrast to prokaryotes, eukaryotic ribosome assembly requires coordinated efforts of the intracellular transport machinery as well as numerous transiently interacting nonribosomal assembly factors. Pre-ribosomal particles released from the nucleolus undergo sequential maturation in the nucleoplasm and cytoplasm before they acquire translation competence. Pioneering work from the Planta and Warner laboratories, in the early 1970s, led to the identification of the earliest preribosome: the 90S that is the common precursor of the mature 60S and 40S subunits. The 90S was thought to contain pre-rRNAs, r-proteins, and numerous assembly factors. 

Assembly of the earliest ribosomal precursor, the 90S pre-ribosome
The process of ribosome assembly in budding yeast begins with RNA polymerase I-driven transcription of ribosomal DNA (rDNA) repeats on chromosome XII in the nucleolus to produce 35S pre-rRNAs (Fig. 1)


Fig. 1 Model for the hierarchical assembly of the 90S pre-ribosome. 
a) The assembly of the tUTP sub-complex is responsible for the initial formation of the 90S. This step allows subsequent incorporation of the indicated sub-complexes. Two independent assembly steps guide 90S formation: recruitment of the U3 snoRNP and UTP-B sub-complexes (top). These primary steps are necessary for the assembly of at least 20 components of the particle. GTPase Bms1 is necessary for a secondary assembly step that promotes the subsequent incorporation of numerous proteins and the Mpp10 sub-complex. A second assembly step involves the incorporation of Rrp5 (bottom), which is crucial for the recruitment of the UTP-C sub-complex but not the U3 snoRNP, the tUTP, or the UTP-B complexes. 
b) Schematic representation of the protein constituents of the 90S subcomplexes. The domain identification was performed using the online tool Pfam

Co-transcriptional association with small nucleolar RNAs (snoRNAs), assembly factors, and r-proteins mainly of the 40S drives the formation of the earliest ribosomal precursor, the 90S pre-ribosome. The emerging 35S pre-rRNA can be cleaved co-transcriptionally in the internal transcribed spacer 1 (ITS1), thereby releasing the pre-40S subunit. Two independent studies have revealed the composition of the 90S that remained refractory to biochemical analyses for nearly 30 years. By isolating Mpp10-TAP, a factor associated with the box C/D U3 snoRNA and a crucial component of the 90S, the Baserga group discovered 17 novel assembly factors, called UTPs (1–17) (Fig. 1). They designated this large >2.2 MDa U3 snoRNA-containing particle responsible for processing of the small subunit (SSU) processome. The composition of the Mpp10-TAP particle significantly overlaps with several nucleolar pre-ribosomal particles isolated and characterized by the Hurt laboratory. Assembly of the 90S appears to be a hierarchical addition of pre-formed protein sub-complexes (Fig. 1a). The stepwise assembly of UTP complexes and the U3 snoRNP is closely coupled to the pre-rRNA modification/folding and possibly drives compaction of the 90S. Several components of the 90S contain motifs involved in RNA-binding and/or protein-protein interaction (Table 1 and Fig. 1b). A protein-protein interaction map of the SSU processome,provides an important framework for the elucidation of the architecture of the 90S. A detailed understanding of protein-protein and RNA-protein interactions will also be crucial to uncover the spatial-temporal assembly of the 90S and its subsequent disassembly. More than 60 different snoRNPs mediate >100 covalent modifications of the 35S pre-rRNA during the assembly of the 90S. There are two types of modifications: methylation of the 2′-hydroxyl group of the ribose sugars (2′-O-methylation) carried out by C/D box containing snoRNAs and conversion of uridine to pseudouridine carried out by H/ACA snoRNAs. These prerRNA modifications by snoRNAs were suggested to be important for correct folding and possibly compaction of the 90S structure. Modifications of the 35S pre-rRNA are accompanied by early pre-rRNA cleavages within the developing 90S pre-ribosome. Binding of U3 snoRNP to pre-rRNA is necessary for the earliest pre-rRNA cleavages at the A0 and A1 sites and crucial for the assembly of the early pre-40S subunit.

Cleavage at the A2 site, by a yet unidentified endonuclease, releases the earliest pre-40S and pre-60S particles (Fig. 2). 



Fig. 2 The assembly pathway of the eukaryotic ribosome. 
a Co-transcriptional recruitment of small subunit r-proteins and assembly factors to the 35S pre-rRNAyields the earliest ribosomal precursor, the 90S (orange). Cleavage at the A2 site separates the 90S into a pre-40S subunit (green) and a pre-60S subunit (blue), which undergo independent maturation. Transiently associating assembly factors drive maturation of the pre-ribosomal subunits as they travel through the nucleoplasm toward the NPCs. Final maturation in the cytoplasm yields translation competent ribosomal subunits. 
b The pre-rRNA processing pathway of preribosomal particles. The common 35S rRNA precursor is trimmed at both ends and cleaved at the A2 site. The cleavage yields 20S and 27SA2 rRNAs which mature independently: The 20S rRNA is processed to 18S rRNA in the cytoplasm. The 27SA2 pre-rRNA is processed in two ways generating two different 5.8S rRNAs. Following the cleavage at the A3 site, the 5′ end of 5.8S is rapidly processed to site B1S, by exonucleases Rrp17 and the Rat1-Rai1 heterodimer. 27SB1L and the 27SB1S pre-RNAs are cleaved at the C2 site, to produce the 7SL/S and the 25.5S pre-rRNA. The latter is converted into the 25S rRNA by Rat1- Rai1 in the nucleus. The 7S pre-rRNAs are processed at the 3′ ends to shorter intermediates and then to 6S1S/1L by the nuclear exosome. Processing of 6S pre-rRNA tomature 5.8S rRNA takes place in the cytoplasm and requires the nucleases Rex1, Rex2, and Ngl2. The 5S rRNA is processed in by the nucleases Rex1, Rex2, and Rex3. 

Pre-40S subunits containing immature 20S pre-rRNAs are rapidly exported into the cytoplasm where they undergo final pre-rRNA processing to 18S rRNA. In contrast, the 27SA2 pre-rRNA precursor within a pre-60S subunit undergoes a series of sequential processing steps to yield the mature 25S rRNA and 5.8S rRNA (Fig. 2b). A third rRNA species of the 60S subunit, the 5S rRNA, is generated by RNA polymerase III (Fig. 2). Processing of the 5S rRNA 3′ end can be carried out by multiple redundant exonucleases Rex1, Rex2, and Rex3. A rationally guided screen for ribosome biogenesis factors uncovered a requirement of Snu66, a component of the splicing machinery, for proper processing of the 5S rRNA precursor. However, its precise contribution to this processing step remains unclear.

Compositional dynamics of pre-ribosomal particles before nuclear export
After separation of the 90S into pre-40S and pre-60S particles, the two precursors follow independent maturation pathways. Pre-40S subunits undergo few compositional changes as they travel through the nucleoplasm and are rapidly exported into the cytoplasm. During their transit through the nucleoplasm, pre-40S subunits associate with protein kinases a (Rio1 and Hrr25) and the ATPase Rio2. These energy-consuming steps possibly prepare the pre-40S subunit for nuclear export and/or final cytoplasmic maturation. However, the relevant substrates for Rio1, Rio2, and Hrr25 remain to be uncovered. The assembly factors Enp1, Ltv1, and the r-protein uS3 (Rps3 form a sub-complex at the landmark beak structure of the 40S subunit. This complex can be dissociated in vitro from pre- 40S subunits by the activity of the conserved kinase Hrr25. It was proposed that phosphorylation of the Enp1-Ltv1-uS3 (Enp1-Ltv1-Rps3) sub-complex increases the conformational flexibility in the head region of the pre-40S subunit in vivo. Hrr25 depletion impairs nuclear export of pre-40S subunits supporting the notion that its kinase activity makes the rigid head region more flexible during transport through the nuclear pore complex (NPC). Dephosphorylation by an unknown phosphatase in the cytoplasm could permit the stable incorporation of uS3 (Rps3) into the mature 40S subunit and formation of the final beak structure. In the 60S assembly pathway, incorporation of 5S rRNA in complex with r-proteins uL18 (Rpl5) and uL5 (Rpl11) is an important early nucleolar/nuclear event. The r-proteins uL18 (Rpl5) and uL5 (Rpl11) are co-imported by Syo1, an adaptor for the importin Kap104. In the nucleus, the uL18-uL5-Syo1 (Rpl5-Rpl11-Syo1) complex is released from Kap104 in a RanGTP-dependent manner and then loaded on the 5S rRNA. Incorporation of uL18- uL5-5S rRNA complex into the pre-60S particle is facilitated by the assembly factors Rpf2 and Rpf1. However, the exact timing of incorporation into pre-60S subunits remains to be investigated. During the journey through the nucleoplasm, pre-60S particles associate with >150 assembly factors as they travel through the nucleoplasm toward the nuclear periphery. At distinct maturation stages, assembly factors are released from pre-ribosomal particles and recycled back to participate in new rounds of biogenesis steps. 

My comment: It is pretty remarkable that over 150 assembly particles accompany the travelling of just one pre-ribosome particle from one compartment to the next. That requires elaborate accurate planning of how to help in the transport by these assembly factors.  How would/could non-goal-directed processes "know" that assembly factors should/could be re-used in various rounds to help the assembly of ribosomes? The re-use of tools is a huge advantage and economy of energy expendure, and evidences elaborate and intelligent engineering principles. 


a–d, Cryo-EM maps of the four structures of NMD3-particles from pre-A to C, gaussian filtered with sDev of 1.058 Å in ChimeraX70. Individual assembly factors, H38, H89, L1 stalk, eL24, eL40 and uL1 are color-coded. Below each map, the overall resolution is shown.

This sequential reduction in complexity of the pre-60S subunits is very likely triggered by a multitude of energy-consuming enzymes. ATP-dependent RNA helicases, AAA-ATPases, ABC-ATPases, and GTPases associate with maturing pre-ribosomal particles and confer directionality to the assembly and maturation process. There are various binding site(s) of these energy-consuming enzymes on maturing pre-60S particles.

a A protein kinase is a kinase which selectively modifies other proteins by covalently adding phosphates to them as opposed to kinases which modify lipids, carbohydrates, or other molecules.

This sequential reduction in complexity of the pre-60S subunits is very likely triggered by a multitude of energy-consuming enzymes. ATP-dependent RNA helicases, AAA-ATPases, ABC-ATPases, and GTPases associate with maturing pre-ribosomal particles and confer directionality to the assembly and maturation process. Three essential AAA-ATPases contribute to pre-60S subunit maturation. The AAA-ATPases Rix7 and Drg1 are closely related to the well-characterized Cdc48 (p97 in mammals) and contribute to early nucleolar and cytoplasmic pre-60S subunit maturation, respectively. Rea1, which is the largest protein in yeast, shares similarity to the microtubule motor protein dynein heavy chain and functions at different nuclear steps during 60S maturation. Rix7 is the first AAA-ATPase that is involved in the maturation of the pre-60S subunit. Like the archetypical AAA-ATPase Cdc48, which uses cycles of ATP hydrolysis to remodel protein complexes, Rix7 was suggested to strip off the assembly factor Nsa1 and facilitate the nucleolar to nucleoplasmic transition of pre-60S subunits along the assembly pathway. It was suggested that Rix7 might recognize posttranslational modifications like ubiquitin and SUMO, either directly or via adaptor proteins, to release Nsa1 and further assembly factors. Rea1 consists of six ATPase modules, forming a binding platform on the ribosome and binds in close proximity to the Rix1-Ipi3-Ipi1 sub-complex. A long α-helical linker domain, a D/E-rich region, and a functionally important metal-iondependent adhesion site (MIDAS) domain follow the ATPase modules. By employing the MIDAS domain, Rea1 can directly contact the MIDAS interacting domains (MIDO) of Ytm1 and Rsa4 and triggers the release of both assembly factors via ATP hydrolysis cycles. Removal of the Ytm1-Erb1-Nop7 sub-complex may trigger the release of neighboring biogenesis factors on the pre-ribosomal particles, as well as recruitment of further assembly factors involved in later biogenesis steps. The release of Rsa4 occurs at a later stage of the assembly pathway and signals the progression toward export competence. Recently, Rea1 and the GTPase Nug2 were implicated in a nuclear checkpoint step that prevents premature formation of an export competent pre-60S subunit. The GTPase Nug2 (also known as Nog2) binds the pre-60S subunits in the nucleus at a site, which clashes with the binding site for the export adaptor Nmd3. Nug2 bound to the pre-60S subunit allows nucleoplasmic maturation to occur. Release of Nug2 depends on its GTPase activity as well as the ATPase activity of Rea1. Only after the release of Nug2, Nmd3 can bind pre-60S subunits and they can be exported. This mechanism provides a timed acquisition of export competence for the large ribosomal subunit.

Nuclear export of pre-ribosomal particles
Assembly of eukaryotic ribosomes begins in the nucleolus, but translation of mRNA into proteins by the mature ribosome occurs in the cytoplasm. Inevitably, pre-ribosomal particles need to be transported through the NPCs into the cytoplasm. NPCs are huge protein assemblies embedded within the double lipid bilayer of the nuclear envelope and serve as ports to exchange macromolecules between the nucleus and cytoplasm. The NPC transport channel permits free diffusion of molecules <40 kDa. Translocation of complex cargos such as the charged >2 MDa pre-ribosome through the hydrophobic phenylalanine-glycine (FG)-repeat meshwork of the NPC channel poses a major challenge. Transport of pre ribosomal particles is facilitated by multiple factors that interact with the FG-meshwork of the NPC channel (Fig. 3). 


Fig. 3 Transport of pre-ribosomal particles through the NPC channel.
Export of the pre-ribosomal particles is facilitated by indicated export factors (yellow) that interact with the FG meshwork of the NPC channel. Cryo-EM maps of late pre-ribosomal particles isolated by Rio2-TAP and Alb1-TAP. 

In actively growing budding yeast cells, it is estimated that each NPC contributes to the export of ~25 pre-ribosomal particles per minute. Such a rapid process requires an efficient transport machinery that ensures rapid translocation of preribosomal cargos through the NPC channel. Cell biological tools that monitored the intracellular localization of ribosomal subunits revealed the requirement of several components of the NPC, the Ran GTPase cycle, and the export receptor Xpo1, in the nuclear export of pre-ribosomes. Subsequently, a visual screen and an independent genetic approach uncovered an essential nuclear export signal (NES) containing adaptor Nmd3. Nmd3 forms a complex with Xpo1 in the presence of the GTPase Ran and facilitates export of the bound pre-60S subunit. Efficient translocation of pre-ribosomes through the NPC requires multiple export factors. Notably, the essential general mRNA transport receptor Mex67-Mtr2 and the HEAT-repeat containing protein Rrp12 contribute to export of both pre-ribosomal subunits  (Fig. 3). Mex67, the large subunit of the heterodimer, is composed of an aminoterminal (N) domain, a leucine-rich repeat (LRR) domain, a nuclear transport factor 2 (NTF2)-like middle domain, and a C-terminal ubiquitin associated (UBA)-like domain. Mtr2 shares structural features with NTF2  and heterodimerizes with the NTF2-like middle domain of Mex67. Loops emanating from the NTF2-like domains contribute to pre-60S and pre-40S subunit binding suggesting a versatile common interaction platform on Mex67-Mtr2. A recent study from the Tollervey laboratory revealed crosslinks between Mex67 and the 3′ end of 20S pre-rRNA transcript as well as 5.8S rRNA and multiple regions along the 25S rRNA in the vicinity of 5.8S rRNA supporting the idea that Mex67 interacts with both pre-40S and pre-60S subunits. The NTF2-like domains ofMex67-Mtr2 and the UBA-like domain ofMex67 interact directly with the FGmeshwork and therefore facilitate nuclear export of the bound cargo. Mex67-Mtr2 does not directly rely on the Ran cycle for export and is the only known transport factor that contributes to the export of three major cargos: mRNA, pre-60S, and pre- 40S subunits. It is not understood how theMex67-Mtr2-pools are split to export all three substrates. Understanding the molecular basis of this allocation will reveal how the three export pathways cross talk to deliver required levels ofmRNA and ribosomal subunits in the cytoplasm.

Rrp12 is the second factor that binds both pre-60S and pre- 40S subunits and is required for their nuclear export. Rrp12 contains secondary structural elements called HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and TOR1) repeats, which are found in other RanGTP-dependent export factors, and shown to interact with the FG-meshwork. Identifying mutants of Rrp12 that are impaired in binding to preribosomal particles, FXFG-repeat nucleoporins and RanGTP will clarify the contribution of Rrp12 to pre-ribosome export. Additionally, pre-ribosomal particles employ multiple nonessential, auxiliary factors that can directly bind FG-rich nucleoporins and directly facilitate translocation of preribosomal particles through the NPC channel (Fig. 3). The trans-acting factor Arx1 that localizes to the exit tunnel plays an auxiliary role in pre-60S subunit nuclear export. Arx1 contains a methionine aminopeptidase (MetAP)-like fold, which is present in a family of proteins that remove the N-terminal methionine from nascent polypeptides as they emerge from the ribosome. However, Arx1 lacks a methionine aminopeptidase activity. Mutations in the methionine-binding pocket impair the function of Arx1 in pre-60S subunit export, which led to the proposal that this fold has evolved to interact with FGnucleoporins. Two recent studies revealed that the proposed FG-interacting pocket of Arx1 points toward the exit tunnel of the 60S subunit and hence does not appear to be exposed to the solvent. Further, Arx1 not only covers the exit tunnel of the 60S subunit, but it also arrests a conserved rRNA expansion segment 27 (ES27) in a so-called tunnel conformation. It could be that Arx1 gets detached from the main body of the pre-60S subunit, while it remains bound to the pre-60S subunit via its interaction with ES27, during translocation of pre-60S subunits through the NPC. Such a scenario may reconcile the apparent paradox, as to how Arx1 simultaneously interacts with the pre-60S subunit as well as FG-rich nucleoporins. Functional screens in yeast have identified factors that directly promote nuclear export of pre-ribosomes. These approaches uncovered the shuttling assembly factors (Ecm1 and Bud20) and mRNA export factor (Npl3) involved in pre-60S subunit nuclear export. Whether pre-ribosomal subunits employ all export factors for their translocation through the NPC is currently unclear. It could be that a minimal set might be sufficient to facilitate rapid export. A particular challenge is to localize export factors on pre-ribosomal particles. These analyses are expected to provide insight into how pre-ribosomal particles are oriented during the translocation through the NPC channel. All export factors described to date utilize the FGmeshwork to translocate pre-ribosomal subunits through the NPCs. Recently, we have uncovered a role for the non-FGinteracting transport factor Gle2 in pre-60S subunit export (Occhipinti et al. 2013). Gle2 interacts with pre-60S through a conserved basic patch and utilizes a second interaction surface to simultaneously bind the GLEBS (Gle2-binding sequence) motif of Nup116 (Fig. 3). These interactions together facilitate the transit of the pre-60S particle through the NPC. Mutations that impair the function of Gle2 in pre-60S nuclear export do not affect mRNA export. Curiously, the recruitment of Gle2 to pre-60S subunits requires its prior tethering to the GLEBS motif of nucleoporin Nup116. Thus, Gle2 could utilize distinct interaction surfaces to prevent kinetic delays experienced by mRNPs and pre-60S subunits during translocation through the NPC channel, especially in the case when cargos have failed to recruit its optimal complement of export factors. In contrast to the pre-60S, fewer export factors are described for the pre-40S subunit. Studies in mammalian cells have revealed multiple NES containing assembly factors (Ltv1, Dim2, and Rio2) that recruit the export factor Crm1 (Xpo1). An essential NES containing adaptor for pre-40S subunits that recruits Xpo1 remains elusive. One possibility could be that multiple NES containing adaptors play redundant roles to recruit the essential export receptor Xpo1 and guarantee efficient nuclear export of pre-40S subunits. Another conserved factor that specifically functions in the nuclear export of pre-40S subunits is the conserved RanGTPbinding protein Yrb2. The yrb2Δ mutant exhibits a marked decrease in the 40S subunit levels and strong nucleoplasmic accumulation of the small subunit reporters. Notably, in vitro studies showed that the human homolog of Yrb2 (RanBP3) triggers the loading of Xpo1 (Crm1) and RanGTP on certain cargoes that are exported into the cytoplasm. Therefore, one possibility could be that Yrb2 delivers Xpo1 and RanGTP to certain yet unknown NES containing adaptor(s) to promote nuclear export of pre-40S subunits. Cytoplasmic maturation pathway of pre-ribosomes Prior to nuclear export, the majority of assembly factors, which associate with pre-ribosomal particles during early biogenesis, are released after fulfilling their function. Only a handful of assembly factors travel with pre-ribosomal particles to the cytoplasm. The release of these factors, the incorporation of the remaining r-proteins, and final prerRNA processing events constitute cytoplasmic maturation in the ribosome biogenesis pathway (Fig. 4). 


Fig. 4 Cytoplasmic maturation of a large pre-ribosomal subunit prior to initiating translation. 
Exported pre-60S subunits are bound by export factors (yellow) and shuttling factors (green) which are released in the cytoplasm. The ATPase Drg1 releases Rlp24 from the pre-ribosomal particles. This event triggers the subsequent maturation events. Arx1 and Alb1 require Rei1, Jjj1, and Ssa1/Ssa2 for their release, whereas the stalk assembly can only take place after the release of the shuttling ribosomal-like protein Mrt4 by the cytoplasmic release factor Yvh1. Recruitment of uL10 (Rpp0) releases Yvh1, which allows further assembly of the P1 (Rpp1) and P2 (Rpp2) heterodimer onto the stalk. Loading of uL16 (Rpl10) triggers the final maturation steps. The GTPase Efl1 and its co-factor Sdo1 release Tif6 and another GTPase Kre35 (Lsg1) removes Nmd3. The release mechanisms/factors of shuttling assembly factors like Nog1, Nug1, and Nsa2 and the transport factorsMex67-Mtr2, Bud20, Ecm1, and Gle2 (depicted in gray) remain to be uncovered

These steps are not only crucial for completing ribosome maturation, but are also crucial for new rounds of ribosome biogenesis. A failure to release and recycle assembly and export factors back to the nucleus induces pre-rRNA processing delays, assembly defects, and impaired nuclear export.

Cytoplasmic maturation of pre-40S subunits
The pre-40S subunits are accompanied to the cytoplasm by a handful of proteins (Enp1, Tsr1, Ltv1, Dim1, Dim2, Nob1, Rio2, and Hrr25) that contribute to their export as well as subsequent pre-rRNA processing. Cytoplasmic processing of immature 20S pre-rRNA within pre-40S subunits involves two conserved events: dimethylation of the 20S pre-rRNA and the endonucleolytic cleavage (site D in Fig. 2b) of 20S prerRNA to 18S rRNA. A late pre-rRNA modification step in 40S biogenesis is the dimethylation of two adenine bases near the 3′ end of the 18S rRNA. The enzyme responsible for catalyzing this modification is the essential factor Dim1 that is loaded on the pre-40S subunits. Dimethylation is first detected on the 20S rRNA precursor and was suggested to take place once the pre-40S particle reaches the cytoplasm. Although dimethylation occurs late during subunit maturation, Dim1 associates already with the 90S. Dim1 depletion causes an early nucleolar pre-rRNA processing defect, which can be rescued by a catalytically inactive dim1 mutant. Intriguingly, the catalytically inactive dim1 mutant rescues the lethality of the dim1Δ strain suggesting that the dimethylating activity of Dim1 is not essential and can be separated from its essential role in early pre-rRNA processing. Dimethylation was suggested to play a role in fine-tuning translation as the dim1 mutant displays increased antibiotic sensitivity. An essential cytoplasmic maturation event that renders pre- 40S particles translation-competent is the endonucleolytic cleavage of the immature 20S rRNA into mature 18S rRNA. Multiple energy-consuming ATPases (Prp43, Rio2, and Fap7) and the PIN-domain endonuclease Nob1 were implicated in this late maturation step. Nob1 is recruited to 40S pre-ribosomes already in the nucleus, suggesting that there must be an activating mechanism for Nob1 in the cytoplasmic compartment. Studies from the Tollervey and Karbstein laboratories revealed that pre-40S subunits interact with mature 60S subunits to form an 80S-like particle in vitro and in vivo. The interaction with the 60S subunit triggers the activity of Nob1 to cleave 20S pre-RNA to mature 18S rRNA in vitro. Both studies implicated that the conserved cytoplasmic GTPase and translation initiation factor eIF5b/Fun12 might be important for the formation of the 80S-like particle, which triggers this final cleavage step in the cytoplasm. Subsequently, Nob1 is released and recycled due to the action of the ABC-ATPase Rli1 that dissociates the pre-40S subunit from the 60S subunit, after 20S pre-rRNA cleavage. Fun12 is not an essential gene in budding yeast, whereas the endonucleolytic cleavage of 20S pre-rRNA is essential. Considering the high abundance of pre-40S subunits, it is intriguing that the fun12Δ mutant shows only minor accumulation of 20S prerRNAs in the cytoplasm. It could be that in a fun12Δmutant, pre-40S subunits containing immature 20S pre-rRNA fail to undergo cytoplasmic maturation and are then rapidly degraded. In support of an 80S-like translation cycle, mutations in the 60S subunit r-protein uL3 (Rpl3), which affect the affinity to translation elongation factors, were shown to specifically impair cytoplasmic 20S pre-rRNA processing.

Cytoplasmic maturation of pre-60S subunits
In addition to shuttling export factors (Nmd3, Arx1, Ecm1, and Mex67-Mtr2), genetic approaches identified few assembly factors (Rlp24, Tif6, Nog1, and Alb1) that associate with pre-60S particles in the nucleus and are transported to the cytoplasm. Release of these factors is catalyzed by conserved energy-consuming GTPases (Kre35, Efl1), ATPases (Drg1, Hsp70), and their cofactors (Sdo1, Rei1, Jjj1) that transiently associate with pre-60S particles exclusively in the cytoplasm. The Johnson laboratory established the order of known release events that provided an initial framework of the cytoplasmic maturation pathway (Fig. 4) (Lo et al. 2010). A characteristic feature of the pre-60S cytoplasmic maturation is the sequential release of assembly factors and transport factors (Fig. 4 and Table 2). The AAA-ATPase Drg1 triggers the earliest cytoplasmic maturation step on pre-60S particles, which is a prerequisite for subsequent cytoplasmic maturation steps of 60S pre-ribosomes. Catalytically impaired drg1 mutants accumulate the nuclear assembly factors Rlp24, Nog1, Arx1, and Tif6 in the cytoplasm where they remain bound to pre-60S subunits. Thus, the activity of Drg1 is required for their release from pre-60S particles, thereby also allowing their reimport into the nucleus. AAA-ATPases typically do not exhibit broad substrate specificities, and therefore, it is unlikely that Drg1 releases each of these factors from pre-60S subunits. The Bergler laboratory demonstrated that Drg1 catalyzes the release of Rlp24 that is essential for subsequent release events. A C-terminal region within Rlp24 acts as a recruiting site for Drg1 to stimulate its ATPase activity. The release of Rlp24 from the pre-60S subunit appears to require the nonessential FG-nucleoporin Nup116. Thus, the mechanochemical activity of Drg1 makes the export step irreversible and simultaneously initiates the cytoplasmic maturation cascade. Release of the ribosomal-like protein Rlp24 is necessary to allow the r-protein eL24 (Rpl24) to assemble into the pre-60S subunit. This exchange event triggers recruitment of the zincfinger proteins Rei1 and Yvh. Rei1 is nonessential, but needed for the recycling of Arx1 and its interacting partner Alb1. Rei1 works in conjunction with the DnaJ domain-containing Jjj1 and the ATPase Ssa1/Ssa2 (Hsp70) to release Arx1. This data also indicates that Arx1 appears to have an inhibitory role in driving cytoplasmic maturation pathway of pre-60S subunits. Based on similarity of Arx1 to MetAPs, a prediction is that they bind to the same site on the ribosome and that Arx1 prevents the binding of MetAP. Further, Arx1 binds in the vicinity of the r-protein uL23 (Rpl25) at the polypeptide exit tunnel, which is an important functional site on the ribosome, as uL23 (Rpl25) interacts with the signal recognition particle (SRP) as well as the translocon in the endoplasmic reticulum (ER). The Johnson and Panse laboratories have uncovered a cytoplasmic maturation event that is crucial for assembly of the ribosome stalk, a structural landmark of the 60S subunit. Assembly of the stalk is a major step in acquisition of functionality of the ribosome, since it is essential for recruitment and activation of translation factors, in particular the elongation factors. In yeast, the stalk is composed of uL10 (Rpp0) and two heterodimers of P1 and P2 (Rpp1/Rpp2). uL10 (Rpp0) anchors the stalk to the ribosome by binding to the rRNA of helices 43 and 44. However, pre-60S subunits are first assembled in the nucleus with the ribosomal-like protein Mrt4 in place of uL10 (Rpp0). Mrt4 lacks the domains that recruit translation factors, necessitating the exchange of Mrt4 for uL10 (Rpp0). The dual specificity phosphatase Yvh1 is required for the removal of Mrt4 and uses its zinc-binding domain but not its phosphatase activity to release of Mrt4 from pre-60S subunits. While the key players are identified, the precise mechanism of Mrt4 release and the molecular events that lead to the assembly of the stalk remain elusive. Following the assembly of the stalk and the removal of Arx1, Tif6 is released. Tif6 is a shuttling assembly factor that prevents the joining of immature 60S to 40S subunits. The release of Tif6 depends on previous events as it mislocalizes in yvh1 mutants in which stalk assembly is blocked. During translation, the stalk functions in recruitment and activation of the GTPase eEF2. Given that Efl1 is closely related to eEF2, stalk assembly might play a similar role in biogenesis, thus recruiting Efl1 for the release of Tif6. This also suggests that the cytoplasmic maturation events in the 60S biogenesis are coupled and ordered sequentially from Drg1-dependent release of Rlp24 to Efl1-mediated release of Tif6 (Fig. 4). The GTPase Efl1 and the Shwachman-Bodian-Diamond syndrome protein Sdo1 are required to release Tif6. In efl1 and sdo1 mutants, Tif6 accumulates on late pre-60S subunits and is mislocalized to the cytoplasm. Mutations in Tif6 that weaken its affinity for the 60S subunit suppress the growthdefects of efl1 and sdo1 mutants, providing strong genetic evidence that Tif6 is the primary substrate of Efl1 and Sdo1. Efl1 bears a significant sequence similarity to the translation elongation factor 2, a GTPase that facilitates translocation of the ribosome following the action of the peptidyl transferase. The essential NES containing adaptor Nmd3 must be released from pre-60S subunits and recycled back to the nucleus. The r-protein uL16 (Rpl10) and the GTPase Kre35 (Lsg1) were implicated in the release of Nmd3. Mutations in uL16 (Rpl10) prevent the release of Nmd3 from pre-60S subunits. Moreover, mutations in Kre35 that are predicted to disrupt its GTPase activity also block Nmd3 release in the cytoplasm. These results suggest that Kre35 triggers the binding of uL16 (Rpl10) to the 60S, an event that is coupled to the release of Nmd3 (Hedges et al. 2005; Karl et al. 1999; West et al. 2005). Interestingly, recent work from the Johnson laboratory indicates that loading of uL16 (Rpl10) on late pre-60S subunits is a prerequisite for the release of Tif6 (Bussiere et al. 2012). Recently, we have employed a combination of genetic trapping, affinity purification, and a targeted proteomic approach based on selected reaction monitoring mass spectrometry (SRM-MS) to interrogate the proteome of 60S preribosomes after nuclear export. Using a resource of SRM assays, we uncovered unanticipated assembly factors (Bud20, Nug1, Nsa2, and Rli1). They are exported to the cytoplasm and are only released after Drg1-mediated release of Rlp24. The functional significance of shuttling behavior of the identified assembly factors is unknown. It could be that they participate directly in their transport and/ or final functional proofreading of pre-60S subunits. Mechanisms that trigger the release of these shuttling assembly factors from pre-60S particles in the cytoplasm remain to be discovered.

Cytoplasmic proofreading systems for the ribosome
Given the importance to correctly translate proteins, an efficient quality control system must ensure that only functional ribosomes enter translation. In the nucleus, the TRAMP complex marks and targets aberrant pre-rRNAs for degradation by the nuclear exosome. Improperly assembled pre-ribosomal subunits that escape nuclear surveillance mechanisms are segregated and targeted for degradation in the cytoplasm by nonfunctional RNA decay (NRD). In the cytoplasm, two antagonistic mechanisms appear to proofread pre-ribosomes: 

(1) Assembly factors might either prevent and/or delay pre-ribosomes from prematurely interacting or initiating translation. 
(2) They could actively check pre-ribosomal subunits for functionality. 

The ribosomal-like proteins (Rlp24 and Mrt4) act as placeholders for r-proteins. Therefore they delay maturation of the subunit and perhaps provide a time window for the functional proofreading of the 60S subunits. The assembly factor Tif6 is another example, whose binding to the 60S subunit interface prevents premature interactions with the 40S subunit. Tif6 is released from pre-60S subunits in the cytoplasm only after the formation of the acidic ribosomal stalk. Efl1 and Sdo1 promote the release of Tif6 from pre-60S ribosomes. Efl1 shares sequence similarity with the GTPase elongation factor eEF2. Additionally, Efl1 was proposed to check the integrity of the GTPase activating center, the P-site of the ribosome for functionality. Thus, cytoplasmic release factors may couple the recycling of shuttling assembly factors and simultaneously check ribosome function. In the case of the pre-40S subunit, a number of assembly factors were proposed to block premature binding of initiation factors, mRNA, tRNA, and the 60S subunit. The cryo-EM structure of a late cytoplasmic pre-40S particle assigned the binding sites for Ltv1, Enp1, Rio2, Tsr1, Dim1, Pno1, and Nob1 on the small subunit ( Fig. 3). Their binding sites imply possible functions in preventing premature translation initiation by blocking access of translation factors. Ltv1 and Enp1 directly bind uS3 (Rps3) on its solvent exposed side thereby blocking the mRNA channel opening. Rio2, Tsr1, and Dim1 bind the subunit interface, thus preventing joining of the mature 60S subunit and translation initiation factor eIF1A. Nob1 and Pno1 block the binding of eIF3 and thereby interfere with translation initiation. After the release of Rio2, Tsr1, and Dim1, such a pre-40S particle may structurally mimic the translation initiation surface of a mature 40S subunit and hence can interact with a mature 60S subunit to form an 80S-like particle. This translation-like interaction could test the ability of a pre-40S subunit to bind 60S subunits. Additionally, by constantly interacting with each other in the cytoplasm, ribosomal subunits may sense their decoding ability to segregate and target aberrant particles for disassembly and degradation. Interestingly, Nob1 activity is stimulated by the translation initiation factor Fun12 (eIF5B). Thus, processing of the 20S pre-rRNA may represent a quality control mechanism that simultaneously triggers subunit maturation and senses translation competence.

Ribosomal RNA Processing and Ribosome Biogenesis in Eukaryotes August 2004
https://sci-hub.ren/10.1080/15216540400010867
In eukaryotes nearly 500 rRNAs, ribosomal proteins, snoRNAs and trans-acting factors contribute to ribosome biogenesis. After more than 30 years of intense research, the incredible complexities of nucleolar function are revealed. The biosynthesis of ribosomal RNA and its incorporation into ribosomes is a remarkably complex process which has been the subject of intense research for more than three decades. Consistent with an ‘RNA-base machine’, ribosome biogenesis begins in the nucleolus with the synthesis of large primary RNA transcripts by RNA polymerase I (Pol I). Initially this nascent pre rRNA is assembled into an 80 – 90S nucleolar particle. Structural rearrangement and nucleotide modifications occur as the ribosomal proteins are incorporated, followed with cleavages which ultimately give rise to the mature ribosomal subunits.  In addition to the 80 or more ribosomal proteins, a surprisingly large number (>150) of accessory proteins and dozens of snoRNAs are involved. The list continues to grow as global approaches are beginning to be applied. 

QUALITY CONTROL IN RIBOSOME BIOGENESIS 
Since the maturation pathway of ribosomes generally is viewed as a collection of many individual steps, at first approximation, these might be expected to proceed independently and some even co-transcriptionally. Indeed, studies in S. cerevisiae have reported an independent maturation of the ribosomal subunits  and, under special circumstances, ribosomes can be synthesized in ‘trans’ with the subunit RNAs expressed from alternate plasmid-associated templates. Such observations raise questions about the need for the nucleolar 80 – 90S ribonucleoprotein particle and why rRNA transcripts are first completed and largely modified before being cleaved. In a competitive environment where about half of the rRNA is derived from plasmidassociated transcriptional units, changes in 3’ ETS structure can completely inhibit not only the removal of the 3’ ETS but also the processing of ITS2, located some 3000 nucleotides upstream, and even depress the production of plasmid-derived 18S rRNA. Similar mutations in the ITS2 can not only inhibit the maturation of the 5.8S and 25S rRNAs but, again, reduce levels of plasmid-derived 18S RNA (71) to 15 – 20% of normal. Mutations in the 5’ ETS which inhibit production of 18S rRNA also can severely inhibit the yield of plasmid-derived 5.8S and 25S rRNAs and even the removal of termination signals can adversely affect rRNA production. Taken together, these observations have been interpreted as evidence of a ‘quality control’ function in ribosome biogenesis; the interdependency in rRNA processing acts, at least in part, as a mechanism which helps assure that only functional RNA is incorporated. The need for such a mechanism actually may reflect an adaptation to an important characteristic of protein synthesis. Since most proteins are made on polyribosomes, a single defective ribosome not only produces less protein but also can inhibit the movement of other ribosomes with much more severe effects on protein synthesis. In a competitive world this greatly amplified effect may make it especially important for cells to avoid forming defective ribosomes, even if infrequent. Figure 2 suggests a model for the assembly of nucleolar preribosome particles which attempts to explain the distant interactions in rRNA processing and their role as a quality control mechanism. 


Figure 2. A model for the assembly of a nucleolar 80 – 90S pre-ribosomal particle.

While ribosomal proteins are assembled on the mature rRNA sequences, nucleolar constituents simultaneously assemble on the spacer regions, forming a nucleolar pre-ribosome particle with three structural domains, two corresponding to pre-ribosomal subunits and a third, real or virtual domain, composed of spacer elements, nucleolar proteins and snoRNAs essential for effective maturation. This process of fitting everything correctly into a large particle may act as a kind of ‘checklist’ to ensure that all is normal. Failing this check makes the faulty pre-rRNA susceptible to ‘housekeeping’ enzymes which rapidly degrade the nascent RNA and prevent its incorporation into ribosomes as observed with many structural mutations. In search of direct evidence for a ‘processing’ domain and structural features which underlie the interdependencies in rRNA processing, attempts have been made to isolate proteins which specifically interact with the individual transcribed spacers. 

Disruptive mutations in the sites affected not only protein binding but inhibited rRNA processing and essentially eliminated the mutated RNA from the mature ribosome population. Because the complex lacked nuclease activity it was tentatively called a ribosome assembly chaperone (RAC), presumably acting as a kind of rack on which critical structure is organized. Evidence for a chaperone function was recently reported when Pac 1 nuclease was observed to direct the complete removal of the 3’ ETS in the presence of RAC protein. Rapid advances in proteomics and global approaches to large RNA/protein complexes through techniques such as TAP are being pioneered successfully and may soon greatly clarify the complex interrelationships which are suggested in Fig. 2. Another mutational analysis in S. pombe recently has raised the possibility that rRNA modification also may act as a quality control mechanism. Many roles have been suggested for rRNA modification, including influences on rRNA conformation, protein binding, rRNA stability and even ribosome function. Despite these suggestions and the complex apparatus for rRNA modification, it is suprising to find that cells lacking most of their snoRNAs or deleted of core snoRNA binding proteins can grow normally. Even pseudouridine residues which appeared to be candidates for a direct role in peptidyl transferase function were not essential. Under competitive conditions, however, substitutions in modified rRNA sequences within the peptidyl transferase center in the S. pombe 25S rRNA render the plasmid-derived mutated RNAs highly unstable and rapidly degraded, also with no effect on cell growth. Since modifications are directed by snoRNAs that share long sequence complementarities with the modified regions and pair with the rRNA to guide the modification process, the interactions could be considered a form of proofreading. Naturally altered sequences would not be expected to pair as effectively and so would tend to disrupt the modification process. In turn, based on the recent experiments in S. pombe, the modified nucleotide position would be unstable or less stable, and at least partially eliminated from the mature ribosome population. As most modified nucleotides are in the active centers of the ribosome, this proofreading would be especially helpful in avoiding deleterious or lethal changes. In summary, we now realize that ribosome biogenesis is even more complex than previously believed and many intriguing questions remain. Nevertheless, new global approaches based on tandem affinity purification, very sensitive mass spectrometry and an assortment of novel probing techniques provide much promise that the complex dynamics of the maturation processes may soon be understood.

Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism 21 June 2019
https://www.nature.com/articles/s41467-019-10678-z
Eukaryotic ribosomes are synthesized in a hierarchical process driven by a plethora of assembly factors, but how maturation events at physically distant sites on pre-ribosomes are coordinated is poorly understood. Ribosomal protein Rps20 orchestrates communication between two multi-step maturation events across the pre-40S subunit. Our study reveals that during pre-40S maturation, formation of essential contacts between Rps20 and Rps3 permits assembly factor Ltv1 to recruit the Hrr25 kinase, thereby promoting Ltv1 phosphorylation. In parallel, a deeply buried Rps20 loop reaches to the opposite pre-40S side, where it stimulates Rio2 ATPase activity. Both cascades converge to the final maturation steps releasing Rio2 and phosphorylated Ltv1. We propose that conformational proofreading exerted via Rps20 constitutes a checkpoint permitting assembly factor release and progression of pre-40S maturation only after completion of all earlier maturation steps.

Introduction
Eukaryotic ribosomes consist of a large 60S and a small 40S subunit, each composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). The synthesis of ribosomes is a highly complex process starting with the assembly of pre-rRNA, r-proteins, and ribosome assembly factors (AFs) into pre-ribosomal particles in the nucleolus. In yeast, the first steps in the synthesis of 40S subunits occur within large precursors termed SSU processomes or 90S particles, in which rRNA folding and processing steps, as well as incorporation of r-proteins and AFs, take place. Endonucleolytic cleavage of the pre-rRNA, together with the dissociation of a large number of AFs, then results in the release of a 43S particle, also termed pre-40S particle, which contains a 3′ extended precursor of the mature 18S rRNA (the 20S pre-rRNA), most 40S r-proteins, and only a few AFs. These particles are exported into the cytoplasm, where further 40S maturation events take place. Finally, conversion of the 20S pre-rRNA into the 18S rRNA by the endonuclease Nob1 results in the release of mature, translation-competent 40S subunits.

AFs Tsr1, Pno1, Nob1, Dim1, and Rio2 are located on the intersubunit side of the pre-40S subunit and that Enp1 and Ltv1 are bound on the solvent-exposed side in the area of the beak structure . These early cytoplasmic pre-40S particles undergo a cascade of maturation events, with the two first and presumably rate limiting ones being two different ATP-dependent maturation steps, resulting in dissociation of AFs Rio2 and Ltv.

We suggest that sensing of the correct conformations of both maturation sites, exerted by Rps20, provides a quality control checkpoint, which ensures that release of Ltv1 and Rio2 is only triggered once all necessary earlier maturation steps have been completed.

Discussion
We unraveled the intricate mechanism leading to the release of AFs Ltv1 and Rio2 from pre-40S particles, which is coordinated by the r-protein Rps20 (Fig. 7).


Model of Rps20-mediated coordination of cytoplasmic pre-40S maturation events.
Maturation events on the solvent-exposed and intersubunit side of pre-40S subunits converge into a final maturation event leading to the release of Ltv1 and Rio2 (see “Discussion”)

On the solvent-exposed side of pre-40S particles, the Rps3 N-domain re-orients and forms its contact with Rps20. This conformational re-arrangement not only presumably weakens Ltv1’s interaction with pre-40S particles but also positions Ltv1 in a way that it can efficiently recruit Hrr25. Owing to competition for overlapping binding sites, Hrr25 likely (partially) disrupts the interaction between Ltv1 and Enp1. Hrr25 then phosphorylates Ltv1 and thereby further destabilizes its association. On the intersubunit side, the Rps20 loop stimulates ATP hydrolysis by Rio2. While mutants lacking the Rps20 loop are viable and cold-sensitive, as are catalytic Rio2 mutants, the combination of both is lethal. This suggests that the Rps20 loop has, apart from its requirement for ATP hydrolysis, a second role, possibly by establishing the structural context needed for final Rio2 release. Most outstandingly, all the above described maturation events on the solvent-exposed and on the intersubunit side can take place independently of each other; however, the ultimate release of both Ltv1 and Rio2 is inhibited as soon as the maturation cascade is disrupted on either side. Only the correct positioning of Rps20 on both sides commits the particles to proceed to Ltv1 and Rio2 dissociation. The eventual triggers for release of Rio2 and Ltv1 are not known. However, based on this study and recent data27, we speculate that Rps20 and/or rRNA re-arrangements are involved. The two AFs may either dissociate at the same time or one after the other. Indeed, there is evidence that Rio2 may dissociate after Ltv.

Comparison of recent cryo-EM structures representing different yeast and human pre-40S maturation intermediates with our C1- and C2-S20Δloop pre-40S structures (Figs. 5 and 6) can help to position our particles in a structural pre-40S maturation timeline.


Cryo-electron microscopic (cryo-EM) analysis of Rps20Δloop pre-40S particles.
Surface views of cryo-EM maps of Ltv1-purified wild-type pre-40S particles16 (left panel) and the Rps20Δloop pre-40S particles (central panel: C1-S20Δloop particles; right panel: C2-S20Δloop particles). Assembly factors and r-proteins of interest have been segmented and colored; the arrowhead next to the C2-S20Δloop map indicates missing density in the platform region compared to the other maps


Structural details of C1-S20Δloop and C2-S20Δloop pre-40S particles. 
a Close-up of the atomic model of C1-S20Δloop, as seen from the 60S interface; segmented cryo-electron microscopic (cryo-EM) densities corresponding to Tsr1, Rio2, and Dim1 are shown in magenta, blue, and lime green, respectively. rRNA is in gray, and other r-proteins in pale blue. 
b Same view of the C1-S20Δloop model as in a, superimposed to the model of pre-40S particles purified with Ltv1 as bait (PDB 6EML). Spatial alignment was realized using the Matchmaker option in Chimera, using C1-S20Δloop rRNA as reference. For more clarity, only Tsr1, Rio2, and rRNA of both models are displayed. Color codes are indicated on the panel. Arrows indicate the rotation of the head of C1-S20Δloop particles compared to the PDB 6EML model. 
c, d Close-up of the heads of C1-S20Δloop (c) and C2-S20Δloop pre-40S particles 
d, as seen from the top of the head/beak of the particles (top-left insets indicate direction of view). Segmented cryo-EM densities corresponding to Rps20, Rps3, Enp1, Asc1, and Rps10 are represented in green, red, purple, pale violet, and turquoise, respectively. rRNA is in gray, and other r-proteins in pale blue

Notably, some of the previously reported pre-40S structures lack densities for Rps3 and Rps20, despite biochemical evidence that they are already assembled at this maturation stage. This suggests high flexibility of the region Rps3 and Rps20 are bound to. Both Rps3 and Rps20 are partly visible in our C1-S20Δloop particles, indicating that they are in a maturation stage where Rps3 and Rps20 have already been partly stabilized. Nevertheless, the Rps20 β-strands are not visible and the Rps3 C-domain sticks out of the pre-40S particles. Moreover, the Rps3 N-domain is in a different orientation than in mature 40S subunits, supporting our previous suggestion that the Rps3 N-domain is initially assembled in an incorrect orientation19.

The actual residues responsible for the interaction between the Rps3 N-domain and Rps20 are not resolved in our C1-S20Δloop structure; therefore, it remains subject to speculation whether this contact has already formed even though the Rps3 N-domain has not yet moved into its final position or whether the structural re-orientation of the Rps3 N-domain and contact formation with Rps20 are coupled and have not occurred yet in this population of S20Δloop particles. Importantly, however, the properties of pre-40S particles incapable of forming the Rps3-Rps20 contact clearly differ from Rps20Δloop particles (Fig. 4), for example, in the ability to recruit Hrr25. Interestingly, the pre-40S structure from the Ban laboratory20 shows a maturation stage where Rps20 and the Rps3 N-domain have already been accommodated in their mature position, while the Rps3 C-domain still sticks out of the particles, suggesting that stable integration of the Rps3 C-domain occurs even later than Rps3 N-domain accommodation. The step-wise incorporation of Rps3 is in line with previously published results showing that both Rps3 and Rps20 initially bind only loosely to pre-40S particles and their stable incorporation occurs at a later stage.

Our cryo-EM structural analysis revealed no altered positioning of Rio2 in C1-S20Δloop particles. It is possible that the absence of the Rps20 loop leads to changes in flexible regions that are not resolved in the structure. Alternatively, the transition from the open conformation of Rio2 to the closed, ATP hydrolysis competent state, which is likely impeded in Rps20Δloop particles, might quickly trigger Rio2 ATP hydrolysis followed by its dissociation, a process that presumably is kinetically too fast to be captured by a static method like cryo-EM.

Our biochemical results strongly suggest that there is a communication between the Rps20 loop and Rio2. We suggest two possible scenarios how this could occur: (1) Both the Rps20 loop and Rio2 directly interact with 18S rRNA helix; therefore, communication between the Rps20 loop and Rio2 may occur via h31. Notably, a recent study revealed that Rio2 ATPase activity is inhibited by RNA27. Hence, re-arrangements in, or dependent on the Rps20 loop may reposition inhibitory rRNA elements, thereby relieving the inhibition of Rio2. (2) An alternative possibility would be a direct interaction between the Rps20 loop and Rio2. Indeed, Rio2 also contains an unstructured flexible loop, which has not been resolved in any of the so far published X-ray or cryo-EM structures. However, the last two resolved amino acids, lysine 129 and serine 145, are positioned close enough to the Rps20 loop20 that such a contact would theoretically be possible. Since, however, neither the Rio2 flexible loop nor rRNA helix h31 are visible in our C1-S20Δloop structure due to flexibility, our structure does not provide further evidence for either model.

It is puzzling that, despite the massive effects of Rps20 loop deletion on pre-40S maturation, rps20Δloop cells are viable. This suggests that at least some pre-40S particles can mature into translation-competent 40S subunits. We hypothesize that the second structural class we observed, C2-S20Δloop, comprising ~12% of the particles in the purification, could represent particles that have escaped the blockade posed by the absence of the Rps20 loop. Alternatively, they might represent particles trapped in a dead end due to the inability to release the remaining AFs like Rio2 and Hrr25. In C2-S20Δloop particles, Rps3 and Rps20 are already in their mature position and Rps10 is assembled, while Ltv1 and Enp1 have either partly dissociated or completely left the particle. Moreover, Rio2 and Tsr1 seem to be only loosely attached to these particles. Interestingly, these particles contain a factor X, which was previously also observed in a late intermediate in a series of human pre-40S structures representing different maturation stages. The factor might be Hrr25, which is enriched in Rps20Δloop pre-40S particles purified via Tsr1-TAP (Fig. 4b); the presence of Hrr25 in such late particles is, however, unexpected and needs to be verified in future studies, especially since Hrr25 appears to require Ltv1 for its efficient binding to pre-40S particles (Fig. 1), while it is unclear whether Ltv1 is still present in the C2-S20Δloop subpopulation.

Although cryo-EM analyses gave important insights into early cytoplasmic pre-40S maturation, it has to be mentioned that the picture obtained from these structural investigations remains incomplete. Indeed, no published structures cover the totality of all distinct particles present in the given bait purification, and often, to reach near-atomic resolutions, a majority of particles has to be omitted from 3D reconstructions because of their flexibility or ill-resolved features. In our case, as frequently in cryo-EM studies, our high-resolution maps included ~30% of the purified pre-40S particles. Furthermore, despite the great progress in the field gained in the last few months, cryo-EM analyses have only provided snapshots describing structurally stable states of purified macromolecules. None of the so far published pre-ribosomal structures were able to fully temporally or causally resolve interdependent, multi-step maturation cascades as we have revealed here by a combination of genetic, biochemical, and structural approaches. Thus our study exemplifies that only by combining structural knowledge with in-depth functional analyses it is possible to unravel individual steps of the pre-ribosomal maturation pathway at a mechanistic level.

Several hundred maturation factors participate in ribosome biogenesis, and it has to be an immense logistic challenge to coordinate their action. A consecutive order in which the point of action of each factor is precisely defined would be highly inefficient, as every single factor would need to communicate with many different, remote factors. Communication across the ribosomal subunit at certain checkpoints, as performed by Rps20 in the Rio2/Ltv1 release pathway, is a much more sophisticated way to coordinate the action of ribosome AFs. Interestingly, additional examples lead to the proposal that communication across nascent ribosomal subunits may play a role in coordinating ribosome biogenesis; however, the molecular mechanisms underlying these processes remain subject to future investigations. Moreover, it is likely that similar long-distance communication mechanisms, as described in our study, are also used to ensure proper assembly and functional regulation of other large ribonucleoprotein complexes.

Ribosome assembly coming into focus  2019 Feb 20
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725133/
In this paper, particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles. Protein synthesis in eukaryotes is carried out by the ribosome, which is a large RNA–protein complex consisting of a small and a large subunit. During protein synthesis, decoding of mRNA by the small subunit is coupled with peptide-bond formation by the large subunit. In the model organism Saccharomyces cerevisiae, the small subunit (40S) comprises 33 ribosomal proteins and the 18S ribosomal RNA (rRNA), whereas the large subunit (60S) comprises 46 ribosomal proteins and 3 rRNAs (25S, 5.8S and 5S rRNA). 

Beginning with the first atomic structures of eukaryotic ribosomal complexes, the structural intricacy of this molecular machine has been revealed2–4. Whereas the ribosomal catalytic centres — the decoding site in the small subunit and the peptidyl transferase centre (PTC) in the large subunit — and other ribosomal functional modules and key architectural features, such as the central pseudoknot in the small subunit and the central protuberance, GTPase activating centre (GAC), P0 stalk and polypeptide exit tunnel (PET) in the large subunit, are evolutionarily conserved, eukaryotic ribosomes contain many additional RNA extensions and proteins5,6 (Supplementary Figs 1,2).

The assembly and thus availability of eukaryotic ribosomal subunits is intimately linked to nutrient availability, stress and the cell cycle (reviewed in7). Approximately 200 non-ribosomal factors, including proteins, protein complexes and small nucleolar ribonucleoproteins (snoRNPs), are required for the assembly of the small and large ribosomal subunits (summarized in tables 3 and 4 in REF.. The maturation of pre-rRNAs for both subunits requires endonucleolytic and exonucleolytic cleavage (FIG. 1). Distinct stages of this process take place first in the nucleolus, then in the nucleus and finally in the cytoplasm. Following a general overview of small-subunit assembly (FIG. 2), we provide a cryo-electron microscopy (cryo-EM) depiction of a nucleolar small-subunit intermediate (FIG. 3). Similarly to the illustration of the small subunit in FIG. 2, the arrival and departure of ribosome assembly factors for the large subunit are illustrated with structures of pre-ribosomal particles in FIG. 4 and serve as a general guide for ribosome assembly. We use the yeast nomenclature for ribosomal proteins throughout.

p53 and ribosome biogenesis stress: The essentials 19 August 2014
Cell proliferation and cell growth are two tightly linked processes, as the proliferation program cannot be executed without proper accumulation of cell mass, otherwise endangering the fate of the two daughter cells. It is therefore not surprising that ribosome biogenesis, a key element in cell growth, is regulated by many cell cycle regulators. This regulation is exerted transcriptionally and post-transcriptionally, in conjunction with numerous intrinsic and extrinsic signals. Those signals eventually converge at the nucleolus, the cellular compartment that is not only responsible for executing the ribosome biogenesis program, but also serves as a regulatory hub, responsible for integrating and transmitting multiple stress signals to the omnipotent cell fate gatekeeper, p53. 3

A significant body of evidence accumulated over the last 10–15 years suggests that alterations of one or more steps that control ribosome biogenesis are essential for malignant transformation and progression, as many key tumor suppressors and proto-oncogenes have been found to regulate this process. Among them, c-MYC and the components of the PI3K-mTORC1 signaling pathway are emerging as key regulators of ribosome biogenesis.  So why exactly is a seemingly innocuous process found in the midst of a battleground between powerful positive and negative cellular regulators? The basic explanation is actually rather simple. Cancer is characterized by uncontrolled proliferation of cells, occurring relatively independently of external stimuli. Yet, cell proliferation cannot take place without proper cell growth, namely an increase in cell mass. The increment in cell mass requires extensive protein synthesis, which is dependent on a constant supply of new ribosomes, effectively coupling ribosome biogenesis and protein synthesis to the cell cycle. This is presumably the reason why genes like c-MYC, which control cell cycle progression and DNA synthesis, have  also to coordinate ribosome biogenesis and protein biosynthesis. While c-MYC evolved to promote cell proliferation and growth, both under normal conditions and as a driver of malignancy, tumor suppressors like p53 and ARF, one of the two products of the INK4a locus, inspectors of cell homeostasis and emerged as gatekeepers of both genomic integrity and ribosome biogenesis.

Reflected by the large number of factors that regulate ribosome biogenesis, the construction of new ribosomes is an elaborate, well-coordinated process, and extremely demanding in terms of energy and resources. It requires the activity of all three RNA polymerases, in order to transcribe both the rRNA and the mRNAs encoding about 80 distinct integral ribosomal proteins (RPs) and other accessory proteins. Ribosome biogenesis also impinges heavily upon the translation apparatus and the nuclear import/export machinery.
Within the cell, the nucleolus is the main site of ribosome biogenesis. It is a sub-nuclear compartment where clusters of tandem repeats of rRNA genes are organized into what is known as nucleolar organizing regions (NOR). The rRNA genes are transcribed by RNA polymerase I (PolI) to produce the precursor 47S rRNA with concurrent processing into mature rRNA species, followed by assembly of the rRNA together with RPs to form the 40S and 60S ribosomal subunits. Notably, different steps of the ribosome biogenesis process are misregulated in a variety of human malignancies, including cancer. A growing number of reports uncover a more complicated picture, where altered activity of the ribosome biogenesis machinery is not merely required to support the rapid proliferation of neoplastic cells, but it might also serve as a driving force in malignancy. Thus, the activity of RNA PolI is deregulated in cancer and other human pathologies, and enhanced rRNA transcription might attenuate the activity of tumor suppressor genes. In addition to excessive RNA PolI activity, many RPs are over expressed in human tumors such as colorectal cancer, esophagus cancer and hepatocellular carcinoma. Cancer cells might benefit from the dysregulation of specific RPs expression, as this might alter quality or quantity of the synthesized tumor promoting proteins or even provide some non-ribosomal advantageous features.


An overview of ribosome biogenesis, both under normal and stress conditions. 
(A) Under basal conditions, rRNA is transcribed and processed in the nucleolus. RPs are imported into the nucleolus, where they are assembled together with processed rRNA into the large and small ribosomal subunits (60S and 40S, respectively). Mdm2 binds p53 and polyubiquitylates it, sending it to proteasomal degradation, possibly assisted by nucleolar-mediated export.
(B) Inhibition of different steps in ribosome biogenesis can cause unassembled RPs and 5S rRNA to bind Mdm2 and prevent p53 degradation.

Remarkably, alongside its role as the hub of ribosome biogenesis, the nucleolus is also a highly sensitive regulatory hub, which is able to sense various stress signals and initiate a plethora of signaling cascades. Of particular interest is a newly recognized signaling pathway involving ribosomal proteins RPL11 and RPL5 as well as 5S rRNA, which has a unique role in conveying stress messages upon impairment of ribosome biogenesis directly to the Mdm2/p53 module. 

The nucleolus as a stress sensor

Because the process of ribosome biogenesis is extremely demanding in terms of energy and resources, its fidelity is closely inspected and virtually any type of severe cellular stress will result in an immediate shutdown of rRNA transcription (Fig. B). In response to such stress conditions, including exposure to different genotoxic agents like doxorubicin or inhibition of rRNA transcription using low levels of Actinomycin D (ActD), the nucleolus undergoes distinct structural changes, including condensation and segregation into structures called nucleolar caps, composed of nucleolar proteins and RNA. Consequently, detection of such structural alterations might be considered as indicative of severe nucleolar stress. In other cases of milder stress, for example when ribosome biogenesis is suppressed by depletion of Rps6, nucleolar morphology is not distinctly altered. Of note, many types of cellular stress, including Hypoxia, heat shock and growth factor deprivation, were reported to activate p53 by blocking different steps of ribosome biogenesis and inducing nucleolar stress (Fig. B). DNA damage has long been known to activate p53 through a variety of mechanisms, yet a revolutionary concept was proposed by Rubbi and Milner. In a vintage experiment they showed that directed DNA damage, using localized UV irradiation, failed to activate p53 when the nucleolus remained unaffected, leading them to conclude that DNA damage alone cannot activate p53 and that nucleolar disruption is a prerequisite for p53 activation. However, future research will be necessary to uncover the underlying mechanisms by which DNA damage inhibits various steps of ribosome biogenesis and understand how various signaling pathways triggered by this type of stress are coordinated during p53 activation.

The first demonstration of a direct connection between p53-dependent cell cycle arrest and inhibition of ribosome biogenesis was provided by Pestov and colleagues, who showed that expression of a dominant negative form of Bop1, an rRNA maturation factor, blocks rRNA processing and activates p53-dependent growth arrest. Additional experimental models were subsequently employed to examine the activation of p53 following attenuation of ribosome biogenesis at multiple stages, such as by inhibition of rRNA transcription following the knockout of TIF-1A or the transient knockdown of RNA PolI catalytic subunit POLR1A. A p53 response can also be elicited by blocking rRNA processing or ribosome subunit assembly, through the stable or transient knockdown of many RPs or ribosomal accessory proteins. Furthermore, ribosomal biogenesis is regulated not only at the stages of biosynthesis and assembly of its various components. Ribosomal proteins are synthesized in the cytoplasm, imported into the nucleus for assembly in the nucleolus, and then exported back into the cytoplasm as mature ribosomal subunits (Fig. A).  This places substantial demands on the nuclear import and export machineries, particularly in light of the large amounts of ribosome-relevant cargo. Perturbation of nuclear import/export can therefore also elicit ribosomal biogenesis stress. Indeed, creating imbalance in the nuclear import of RPs through the knockdown of a single nuclear import factor, Importin 7 (IPO7), suffices to trigger p53 activation; this activation is dependent on RPL5 and RPL11, confirming that it emerges through bona fide ribosome biogenesis stress (Fig. B).

Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster 2019 Jun; 20 4
Energetically speaking, ribosome biogenesis is by far the most costly process of the cell and, therefore, must be highly regulated in order to avoid unnecessary energy expenditure. Not only must ribosomal RNA (rRNA) synthesis, ribosomal protein (RP) transcription, translation, and nuclear import, as well as ribosome assembly, be tightly controlled, these events must be coordinated with other cellular events, such as cell division and differentiation. In addition, ribosome biogenesis must respond rapidly to environmental cues mediated by internal and cell surface receptors, or stress (oxidative stress, DNA damage, amino acid depletion, etc.). (PI3K-AKT-mTOR, RB-p53, MYC) control ribosome biogenesis and  interact with some of the less well studied pathways (eIF2α kinase and RNA editing/splicing) in higher eukaryotes to regulate ribosome biogenesis, assembly, and protein translation in a dynamic manner.

Ribosome biogenesis is the process by which the 47S and 5S ribosomal RNAs (runes) are transcribed, processed, and assembled with the necessary ribosomal proteins to form the small (40S) and large (60S) ribosomal subunits. Once exported to the cytoplasm, the two subunits join, in the presence of mRNA and initiator tRNA to form the pre-initiation complex (PIC). Further processing results in a mature ribosome. Ribosome biogenesis represents the most expensive, complex, finely tuned, multi-step process that the cell must carry-out; therefore, it happens to be one of the most intricately regulated and controlled. In the case of eukaryotes, the process involves the input of all three RNA polymerases (RNA pol I, RNA pol II, and RNA pol III), 79 ribosomal proteins (33 in the 40S subunit and 46 in the 60S subunit), and well over 200 proteins (helicases, splicing factors, and chaperone proteins) and non-coding RNA (ncRNA) species (miRNAs, scaRNAs, and snoRNAs). 

Ribosome biogenesis in cancer: new players and therapeutic avenues 01 December 2017 5
The ribosome is one of the oldest molecular machines in extant life, and its biogenesis is one of the most complex biological processes

My comment: Do you see the paradox? If naturalistic evolution were true, the progress should be from simple to complex. But here we see a molecular protein factory, a literal 3D Printer, that had to EMERGE PRIOR WHEN LIFE BEGAN, and  " its biogenesis is one of the most complex biological processes".

They continue:

"Despite the strong degree of conservation of their core structure throughout evolution, the ribosomes of higher eukaryotes have the most complex architecture, and an elaborate biogenesis programme, as revealed by high-resolution structural analyses".

My comment: Conserved, in evolutionary parlance means, not evolved any further. So, somehow, for unknown reasons, PRE-darwinian evolutionary mechanisms, made this still today not fully understood superlative nano-machine complex, and then, evolution simply came to a halt. Amazing. Unbelievable. Literally.

The process initiates in the nucleoli and is followed step-by-step with sequential rounds of assembly and modification of the maturing ribonucleoprotein (RNP) complexes as they migrate from the nucleoli to the nucleoplasm and ultimately to the cytoplasm, where the final assembly and maturation steps take place. Mutations in any of the necessary proteins or alterations at practically any of the maturation steps can result in dire consequences to the organism, depending on both the penetrance of the alteration and the tissue involved. Thus, ribosome biogenesis is highly regulated with diverse checkpoints to limit the production of altered ribosomes.

My comment:  This is evidence that the process of ribosome assembly had to emerge fully functional and developed, in order prevent the process to drive havoc and not lead to a functional ribosome machine.

Additionally, the process of ribosome biogenesis is energetically expensive for the cell; its regulation must coincide with the environmental conditions in which the cell finds itself and with other cellular processes, such as cell division and differentiation. Under low nutrient conditions, ribosome biogenesis and protein synthesis would not be energetically favorable to the cell. Similarly, initiating ribosome biogenesis and protein synthesis at the same moment as cell division rather than prior to or following cellular division would be catastrophic to the cell. 

My comment:  That is another hard evidence that this process could not have emerged in a slow developmental stepwise evolutionary process.

Nucleolar Stress: hallmarks, sensing mechanism and diseases  2018 May 10 7
The nucleolus is a prominent subnuclear compartment, where ribosome biosynthesis takes place. Recently, the nucleolus has gained attention for its novel role in the regulation of cellular stress. Nucleolar stress is emerging as a new concept, which is characterized by diverse cellular insult-induced abnormalities in nucleolar structure and function, ultimately leading to activation of p53 or other stress signaling pathways and alterations in cell behavior. Based on literature of the past two decades, we herein summarize the evolution of the concept and provide hallmarks of nucleolar stress. 

The nucleolus is a subnuclear compartment, which is primarily known for its role in ribosome biosynthesis. Within nucleoli, genes for ribosomal RNA (rDNA) are arranged in arrays of tandem repeats a), precursors of ribosomal RNA (rRNA) are transcribed by the RNA polymerase I (Pol I) and processed, before the ribosomal proteins are incorporated and ribosomal subunits are assembled. However, during the past two decades, researchers have demonstrated that this is in fact an organelle having multiple complex functions. Several lines of evidence have revealed the most intriguing novel role of the nucleolus as a sensor for various cellular stresses, eventually leading to the concept of ‘nucleolar stress’. Numerous studies have listed triggers for nucleolar stress, characterized morphological and functional alterations, and dissected the molecules that induce activation of p53 signaling or other stress-responsive pathways. While these studies enriched our understanding of the general features of nucleolar stress, many questions, especially those regarding the sensing mechanism, remain unanswered.

Cell cycle progression depends on some aspect of ribosome biogenesis..Perturbation in ribosome biogenesis may cause nucleolar stress, leading to cell cycle arrest in a p53-dependent manner. Indeed, this model of nucleolar stress, probably the first of its kind, is consistent with many observations under diverse p53-activating stressors. A large variety of cellular stress situations can be integrated by a single molecule, namely p53. A common phenomenon in all p53-inducing stresses is nucleolar disruption. Based on a comparative meta-analysis of diverse stimuli that activate p53 signaling and induce nucleolar alteration, they hypothesized that the impairment of nucleolar function might stabilize p53. In fact, activation of p53 is induced by a wide range of cellular stresses, aside from the Pol I inhibitor Act.D, which all cause disruption of nucleolar organization. The translocation of nucleophosmin (NPM1, or B23), an abundant nucleolar protein that is the most frequently reported to move to the nucleoplasm and cytoplasm upon various cellular insults was set as the criterion for nucleolus disruption.NPM1 translocation, or nucleolus disruption following micropore UV irradiation over the nucleoli occurs prior to and independent of p53 induction. Alternatively, p53 response can be induced by interfering with nucleolar function using an antibody against the nucleolar protein UBF (upstream binding factor) in the absence of any genotoxic insult. Therefore, the model they proposed was the only one that could provide a unifying and coherent explanation for the action of all known p53-stabilizing agents.

Ribosome biogenesis insults and a wide range of stimuli as stressors
Ribosome biogenesis comprises multiple steps accomplished in three distinct subnucleolar components, from Pol I transcription initiation to pre-rRNA processing and ribosomal assembly. Any error that causes disturbance in ribosome biogenesis will lead to nucleolar stress.

In fact, deletion or aberrant expression of a number of ribosomal proteins induce p53 stabilization and activation via disruption of ribosome biogenesis: Perturbation of the nucleolar protein Bop1 activity could induce ribosome biogenesis impairment, followed by a p53-dependent cell cycle arrest. Genetic inactivation of TIF-1A, a basal transcription initiation factor for Pol I, leads to nucleolar disruption, cell cycle arrest and p53-mediated apoptosis. Depletion of importin 7 (IPO7) or exportin 1 (XPO1) proteins impairs ribosome biogenesis and also initiates p53-dependent cell cycle arrest. Microinjection of specific monoclonal antibodies against transcription factor UBF inhibits rRNA transcription and leads to p53 stabilization. Overall, systematic screening analysis revealed an extensive connection of p53 stabilization with nucleolar disruption induced by ribosomal protein depletion.

The chemotherapeutic agent Act.D is the mostly used nucleolar stress inducer. It may inhibit three individual RNA polymerases at different concentrations. It is believed that Act.D can induce DNA damage and inhibit general transcription at high concentrations, such as 430 nM, but selectively inhibits Pol I and induces ribosomal stress at low dose like 5 nM.

Strikingly, stressful conditions that can induce p53 activation can all induce nucleolar stress: these include UV light, hypoxia, heat shock, nucleotide depletion and various chemotherapeutic agents. These stimuli were confirmed to simultaneously induce nucleolar stress and p53 activation by subsequent studies. Common cellular insults that are able to induce p53 activation can also induce the translocation of NPM1, a hallmark of nucleolar stress, in a reactive oxygen species (ROS)-dependent manner. Moreover, our study added nutrient starvation and direct exposure to hydrogen peroxide (H2O2) to the growing list of nucleolar stress inducers.

In summary, the reported factors that induce nucleolar stress can be classified into two categories: canonical and non-canonical. The former points to those affecting homeostasis of ribosome biogenesis, whereas the latter includes a wide range of general cellular insults.  


Stressors eliciting nucleolar stress.
Two categories of nucleolar stress inducers are direct ribotoxic insults and a wide range of cellular insults.

Nucleoplasmic translocation of nucleolar proteins
Unlike membrane-limited organelles, there is no structural barrier between the nucleolus and the surrounding nucleoplasm. As a consequence, any soluble molecule can potentially traffic in and out of the nucleolus in a relatively free manner. This shuttling might occur at basal levels under non-stressful ‘resting’ conditions, but is significantly increased under various stress conditions. Nucleolar stress causes a lot of nucleolar molecules to redistribute in the nucleus, or in other words, to be released from the nucleolus to the nucleoplasm. This translocation or redistribution is thus considered as an indicator of nucleolar stress.

NPM1
NPM1 (also known as B23, nucleophosmin, numatrin or NO38) is the most abundant protein in the nucleolus and under diverse scenarios can dynamically shuttle both within nucleoli and between the nucleolus and the nucleoplasm or the cytoplasm. The known functions of this protein include the interaction with a plethora of macromolecules, for instance, Rb in the nucleus and BAX in the cytoplasm, and chaperoning activity protecting proteins from aggregation in the crowded nucleolar environment. At exit of mitosis, NPM1, among other ribosomal processing proteins, undergoes bidirectional traffic between incipient nucleoli and perinucleolar bodies, which may contribute to nucleolar assembly in early G1 phase. NPM1 is also responsible for the nuclear export of ribosomal protein L5.

Furthermore, a wide range of anticancer agents aside from specific inhibitors also induce NPM1 nucleoplasmic translocation, including the inosine-5'-monophosphate (IMP) dehydrogenase inhibitor tiazofurin, DNA topoisomerase II (topo II) inhibitors doxorubicin and daunomycin, topo I inhibitors mitomycin C and camptothecin, phosphatidylinositol kinase inhibitor toyocamycin and JAK/STAT3 inhibitor cucurbitacin B. Even an iron chelator deferoxamine which showed anti-proliferation effects, UV radiation, viral infection, hypoxia and oxidative stress (H2O2) all lead to nucleoplasmic translocation of NPM1.

Among the observations of nucleoplasmic translocation of NPM1, a great part described the association of this event with p53 signaling activation. There is a relationship between NPM1-translocation and apoptosis. . UV irradiation-induced p53 activation was dependent on NPM1 interaction with HDM2, suggesting that NPM1 activates p53 in a regulated fashion. There is a redox mechanism of NPM1 for sensing nucleolar stress that causes p53 accumulation and activation.

Therefore, as a most frequent event, NPM1 translocation should be regarded as a conspicuous hallmark of nucleolar stress.

Other nucleolar proteins
The following nucleolar proteins exhibit nucleoplasmic translocation under particular types of nucleolar stresses. However, their translocations are not yet explored as universally under many stress conditions as NPM1.

Nucleolin, alias C23, a DNA and RNA binding protein, is essential for pre-RNA transcription, folding, processing and assembly. Cyclin-dependent kinase inhibitor roscovitine induced both nucleolin translocation and nuclear accumulation of p53. Nucleostemin that functions in pre-RNA processing was also translocated to the nucleoplasm under doxorubicin and Act.D treatments in neonatal rat cardiomyocytes, which occurred concurrently with p53 accumulation.

Morphological descriptions for nucleolar stress
According to the classical ‘tripartite’ model, the three main events for ribosome biogenesis, i.e., pre-rRNA transcription, processing, and ribosomal subunit assembly, are reflected in three distinct subnucleolar compartments named the 

fibrillar center (FC), the 
dense fibrillar component (DFC), and the 
granular component (GC). 

It is generally accepted that pre-rRNA is transcribed from rDNA in the FC or at the border between the FC and DFC. FCs are enriched in components of the RNA Pol I machinery, such as UBF, whereas the DFC harbors pre-rRNA processing factors, such as the snoRNAs and snoRNP proteins, fibrillarin and Nop58. Both the FC and the DFC are surrounded by the GC, where pre-ribosome subunit assembly takes place. The morphology and size of nucleoli are linked to nucleolar activity, which are inevitably altered under stress conditions, showing a variety of reorganization.

Nucleolar segregation has emerged as an indicator of nucleolar stress induced by, in particular, agents that cause rDNA damage and rRNA transcription impairment. For instance, chemotherapeutic agents that inhibit rRNA transcription and early processing steps, but not late processing steps, lead to the loss of nucleolar integrity, which is marked by NPM1 translocation to the nucleoplasm. Meanwhile, after chemotherapeutic agent treatment that mostly inhibit early and late rRNA processing steps, fibrillarin and the ribosomal biogenesis factor pescadillo are translocated into distinct morphological subnuclear structures, namely nuclear spots and nucleolar caps structures or even form ‘necklace’ structures (especially for fibrillarin). 

Activation of p53 signaling
The p53 tumor suppressor protein is considered as an integration point in response to various cellular stresses. The activation of p53 can promote transcription of p21 leading to G1/S growth arrest, of 14-3-3 sigma inducing G2/M arrest, or of Bax inducing apoptosis. It can also induce other factors involved in autophagy, DNA repair and metabolism.

The major negative regulator of p53 is the E3 ubiquitin ligase MDM2 (murine double minute 2, HDM2 in human). Mechanistically, MDM2 interacts with p53 via its C-terminal RING finger domain, promoting p53 ubiquitination and degradation by the 26S proteasome. Therefore, p53 stabilization and activation in response to various stresses rely on a disruption this interaction between p53 and MDM2/HDM2. Simple readouts for p53 activation under nucleolar stress conditions are an increased p53 protein levels (stabilization or accumulation following blockage of ubiquitin-proteasomal degradation), reduced p53 binding to MDM2/HDM2, increased p53 mRNA levels under a long-lasting stress, elevated mRNA levels of p53 target genes, typically CDKN1A (p21) and BAX, and corresponding cell phenotypes such as cell cycle arrest, autophagy, DNA repair, senescence, or apoptosis.

Involvement of p53-independent stress signaling
In p53-/- or p53 inactivated cell lines, nucleolar stress can usually still invoke cell cycle arrest or apoptosis, implying that there is a stress response that is mediated by signaling pathways other than p53.

Ribosomal proteins (RPs) regulating transcription factors (TFs)
The major non-p53 TFs that respond to ribosomal stress are c-Myc, E2Fs and SP1. Their downregulation or decreased transcriptional activities by RPs mediate cellular stress responses via altered transcription of target genes. Measurement of mRNA and/or protein levels of these TFs and their target genes, and analysis of TF binding with the target DNA, may indicate the involvement of these signaling pathways.

The oncoprotein c-Myc positively controls cell growth and proliferation and serves as a direct regulator of ribosome biogenesis; many products of its transcriptional target genes are involved in ribosome biogenesis. As a feedback mechanism, RPL5 and RPL11 are two critical negative regulators of c-Myc expression during ribosomal biogenesis; they form a complex with c-Myc mRNA and recruit microRNAs to repress c-Myc expression thus inhibiting the transcriptional activity of c-Myc. RPS14 may also function as a negative regulator of c-Myc. Consistently, upon nucleolar stress, as ribosome-free RPs, these proteins can lead to inhibition of cell proliferation through suppression of c-Myc and its target gene expression 102.

E2F-1 is a member of the E2Fs family of transcription factors; the expression of their target genes are important both for cell proliferation and apoptosis. Independent of its regulatory control of p53, MDM2 prolongs the half-life of E2F-1. Under impaired rRNA biosynthesis, free RPL11 binds to MDM2 causing E2F-1 degradation, which is associated with the inhibition of cell proliferation.

Recently, RPL3 has been found as a pro-apoptotic factor under nucleolar stress induced by 5-fluorouracil in colon cancer cells devoid of p53. RPL3 in ribosome-free form, negatively regulates cystathionine-β-synthase (CBS) expression at the transcriptional level through inhibition of Sp1 binding to the CBS gene. In addition, RPL3 can mediate p53-independent p21 upregulation, which requires the specific interaction between RPL3 and Sp1. Depending on its intracellular levels, p21 can either induce G1/S arrest of the cell cycle or mitochondria-mediated apoptosis.

RPs regulating non-TF proteins
RPL3 can not only negatively regulate CBS expression at the transcriptional level, but also trigger CBS translocation into mitochondria. Consequently, apoptosis is induced through the mitochondrial apoptotic cell death pathway.

Nucleolar proteins regulating TFs and non-TF proteins
There are several nucleolar proteins that bypass p53 and directly promote cell cycle arrest or apoptosis. These p53-independent regulators of apoptosis mainly include NPM1, PPAN, ARF and NuMA. Both NPM1 and ARF are well-known for their roles in p53 signaling, however, several reports have demonstrated their involvement in p53-independent signaling. In these cases, translocation of the nucleolar proteins and their interactions with the corresponding proteins may be analyzed. Interestingly, many RP or other nucleolar protein-mediated p53-independent stress responses require NPM1. In fact, NPM1 alone also interacts with apoptotic proteins. In conditions of nucleolar stress, NPM1 is transcriptionally induced and relocalizes from the nucleolus to the cytoplasm where it complexes with BAX, a crucial effector of the mitochondrial apoptosis pathway. Of note, cytosolic NPM1-BAX interaction has also been associated with cell resistance to death stimuli, therefore, the cellular response this direct interaction of NPM1 with apoptosis regulators does not necessarily result in cell death.

The Wnt target Peter Pan (PPAN) localizes to mitochondria in addition to its nucleolar localization and inhibits the mitochondrial apoptosis pathway in a p53-independent manner. Its role as an anti-apoptotic factor is indicated by the fact that knockdown of PPAN induces BAX stabilization, mitochondrial membrane depolarization and cytochrome c release. Staurosporine or Act.D-induced nucleolar stress and apoptosis disrupt nucleolar PPAN localization and induce its accumulation in the cytoplasm, which might be associated with impairment in its anti-apoptotic function.

Recently, the nuclear mitotic apparatus protein NuMA that locates in nucleoli in the interphase, has been demonstrated to be redistributed upon Act.D or doxorubicin- induced nucleolar stress. NuMA co-immunoprecipitates with Pol I, with RPL26 and RPL24, and with components of an ATP-dependent chromatin remodeling complex associated with rDNA transcription. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53 108.

Several studies reported that ARF binds and antagonizes the transcriptional activities of c-Myc and E2F-1, halting cell cycle progression in absence of p53. In addition to the regulation of the TFs, ARF controls proliferation by limiting nucleolar localization of the RNA helicase DDX5, which ultimately increases ribosome output.

Epigenetic control of ribosome biogenesis homeostasis 2 21 Sep 2018
Ribosome biogenesis is monitored at several stages of cell life
Ribosome biogenesis activity is tightly regulated and can be inhibited a number of ways in times of metabolic distress. Thus, ribosome synthesis can be considered a proxy for cell health. Indeed, several mechanisms monitor the state of ribosome biogenesis and regulate cell fate in accordance. 

Cell cycle commitment 
One such example is the progression into the cell cycle. Indeed, the transition from G1 phase to S phase, implies a commitment to cell division, and is subject to extensive regulation. Passing the G1/S checkpoint requires to reach a critical size, and the same condition applies for the G2/M checkpoint. As ribosomes constitute the largest part of the cell material, their abundance may serve to evaluate cell size. In turn, ribosome number may be measured through translation capacity. In yeast, the depletion of many translation factors and tRNA biosynthesis genes induce an arrest of the cell cycle in G1 phase, suggesting that G1 to S phase transition is translation-dependent. Translation of the yeast Cln3p 8 was shown to be required to pass this checkpoint.

My comment: Passing a checkpoint requires pre-programming and logic. If X, then Y. This is like a logic gate. If the Cln3p gene product is expressed, the checkpoint informs to the cell: We can move on in the Cell cycle.  This is a closed system, where all players have to be in place, in order for this system to work in a coordinated fashion. That is the checkpoint molecule, and the program of analysis and recognition of the situation, and the signaling instructions and network how to move on in either of the cases and react accordingly. The implementation of such a system is clearly evidence of intelligent setup and design.

Cln3p is an extremely unstable protein, so its accumulation requires intense translation. In addition, the presence of an upstream open reading frame (uORF) in the 5’ UTR of the Cln3p mRNA represses its translation when the amount of ribosomes is limited. Thus, accumulation of Cln3p is only possible after ribosome number reaches a certain point. Depletion of an rRNA processing factor triggered defects in cell cycle progression before the number of ribosomes or translation capacity started to dwindle (diminish gradually in size, amount, or strength) , in a Cln3p-independent manner. Ribosome biogenesis activity is monitored at the level of newly synthetized subunits to trigger cell cycle progression. The depletion of r-proteins in yeast caused stage specific cell-cycle arrest. Many of them caused G1 phase arrest, consistent with monitoring of either ribosome biogenesis or steadystate levels. Interestingly, nine r-proteins of the large subunit triggered an arrest in G2 phase, suggesting that they are required at the G2/M checkpoint. This specific defect could result from another mechanism than G1 arrest. Strikingly, all nine r-proteins cluster on the solvent side of the 60S subunit, where they could interact with non-ribosomal factors. 

My comment: This is not only striking but evidence of amazing bioengineering marvel. During ribosome biogenesis, the individual protein strands are located where they are able to communicate (!!!) with surrounding factors and inform: "everything going fine here", or " there is a problem, stop everything". Communication of that kind of sort requires always an irreducible and integrated communication system, that could and would not evolve in a stepwise gradual fashion, because intermediate stages cannot perform the information processing in question. 

Thus, they may be needed either as part of “specialized” ribosomes, or they could participate in cytoplasmic export of G2 phase-specific factors.

Noncoding RNAs in eukaryotic ribosome biogenesis and function 2015 9
The ribosome, central to protein synthesis in all cells, is a complex multicomponent assembly with rRNA at its functional core. During the process of ribosome biogenesis, diverse noncoding RNAs participate in controlling the quantity and quality of this rRNA. The human ribosome contains four rRNAs and 80 ribosomal proteins. Ribosome biogenesis is a highly orchestrated process involving hundreds of molecular components and trans-acting proteins termed assembly factors (AFs). 

What are Assembly Factors?

The concerted action of 76 small nucleolar RNPs (snoRNPs) and ~200 trans-acting proteins, referred to as assembly factors (AFs), facilitates production of translation-competent ribosomal subunits in S. cerevisiae. AFs include proteins with a wide range of biochemical activities, such as endonucleases and exonucleases, GTPases, ATP-dependent RNA helicases, AAA-ATPases, kinases, and structural proteins, predicted to function as scaffolds or chaperones. During ribosome assembly, the structural AFs help to stabilize pre-ribosomes, and to establish molecular interaction networks within pre-ribosomes. The enzymatic AFs link maturation events with substantial changes in free energy to remodel molecular interaction networks during assembly. The structural and functional integrity of pre-ribosomes are tested prior to their entry into the translation pool by AFs 10

In eukaryotes, the process starts with precursor (pre)-rRNA synthesis in the nucleolus, where the synthesized pre-rRNA is modified, folded and processed. These steps are catalyzed with the aid of small nucleolar RNAs (snoRNAs), which are active as part of small nucleolar ribonucleoprotein particles (snoRNPs). All ribosomal components are then assembled and transported to the cytoplasm, and there are qualitycontrol steps throughout.

Ribosome assembly dysfunction leads to ‘ribosome diseases’, or ribosomopathies. These severe human diseases result from mutations in RPs or ribosome assembly factors, and they are characterized by hematological defects, skeletal problems and increased cancer susceptibility. 


Eukaryotic ribosome biogenesis at a glance. 
Ribosome biogenesis encompasses six important steps (yellow boxes): 

(i) transcription of components (rRNAs, mRNAs encoding ribosomal proteins (RPs) and assembly factors (AFs), and snoRNAs); 
(ii) processing (cleavage of pre-rRNAs); 
(iii) modification of pre-RNAs, RPs and AFs; 
(iv) assembly; 
(v) transport (nuclear import of RPs and AFs; pre-ribosome export to the cytoplasm); and 
(vi) quality control and surveillance. 

Three out of four rRNAs are transcribed in the nucleolus by Pol I as a long 47S precursor (47S pre-rRNA), which is then processed and modified to yield the 18S, 5.8S and 28S rRNAs that are assembled into the pre-40S (green) and pre-60S (orange) ribosomal subunits. 5S rRNA (pink) is transcribed by Pol III in the nucleoplasm and incorporated into maturing 60S subunits, forming the central protuberance (CP). 80 RPs, more than 250 AFs and 200 snoRNAs are transcribed by Pol II. The proteins are synthesized in the cytoplasm and reimported to the nucleus for assembly. Pre-40S subunits are exported to the cytoplasm more rapidly than pre-60S subunits, which require numerous nuclear maturation steps. Several structures important for ribosome function are formed only in the cytoplasm, including the beak on the 40S subunit and the stalk on the 60S subunit; both are protruding features that could obstruct subunit export if formed prematurely. Pre-40S subunits undergo a ‘test drive’ to prove functionality before final maturation

Synthesis involves all three RNA polymerases (Pol I–III). In humans, three out of four rRNAs are transcribed in the nucleolus by Pol I as a long 47S precursor. Genes encoding 80 RPs and >250 AFs are transcribed by Pol II. Many snoRNAs are processed from pre-mRNA introns; others are synthesized from their own promoters by Pol II or Pol III. 5S rRNA is transcribed by Pol III in the nucleoplasm. Within the Pol I–transcribed precursors, mature rRNAs are embedded in noncoding spacers: 5′ and 3′ external transcribed spacers (5′ and 3′ ETSs) and internal transcribed spacers 1 and 2 (ITS1 and ITS2). Pre-rRNA processing removes these noncoding spacers
accurately, generating the mature 5′ and 3′ termini of rRNAs. 


Figure 2: Pre-rRNA processing pathways in human cells.
18S, 5.8S and 28S rRNAs are produced from a single RNA Pol I transcript (47S). The mature sequences are embedded in noncoding 5′ and 3′ external transcribed spacers (ETS) and internal transcribed spacers (ITS1 and ITS2). All cleavage sites are marked on the 47S precursor, and cleavage steps are indicated in blue. 47S is cleaved at sites 01 and 02 on both sides of the molecule to generate the 45S pre-rRNA, which is processed by two alternative pathways. In a minor pathway (pathway 1), site A0 and site 1 are cleaved first to yield the 41S pre-rRNA. Uncoupling of processing at sites A0 and 1 leads to the 43S intermediate (red arrows). The 41S pre-rRNA is digested at site 2 to separate 21S and 32S pre-rRNAs, the precursors destined to form the small and large subunit, respectively. 21S pre-rRNA is cleaved at site E to produce the 18S-E intermediate, which is then processed at site 3 into the mature 18S rRNA in the cytoplasm. Processing of the 32S within ITS2 generates the 12S pre-rRNA and the 28S rRNA. The 12S pre-rRNA is successively trimmed to produce the 5.8S rRNA by a series of exoribonucleolytic digestions. There are two forms of 5.8S rRNA, 5.8SS and 5.8SL (5.8L/S), with the latter indicated by the red extension (additional information in Box 3). In the other major pathway (pathway 2), the 45S pre-rRNA is directly cleaved at site 2 to generate the 30S and 32S pre-rRNAs. Processing of the 30S pre-RNA at sites A0 and 1 produces 21S, whereas the 26S pre-rRNA arises from uncoupling at cleavage sites A0 and 1 (red arrows). 21S and 32S processing are similar in both pathways. Additional details are in ref. 26 and at http://www.ribogenesis.com/.




Pre-rRNA processing always starts within the noncoding spacers and never at the mature rRNA ends, and it involves both endo- and exoribonucleolytic digestions. In addition, it imparts directionality to ribosome biogenesis and potentially supplies the energy stored in phosphodiester bonds for structural-remodeling events. Alternative pathways act as backup mechanisms, ensuring robustness. For example, in yeast, the 5′ end of 5.8S rRNA can be generated by either the 5′-3′ exoRNase Rat1–Rai1 assisted by Xrn1 or, in a parallel pathway, by Rrp17.

There are over 200 trans-acting AFs in budding yeast, a reference eukaryotic model organism. These and the many snoRNAs (75 in yeast, ~200 in humans) required to assemble ribosomes make the ribosome-assembly machinery far more complex than the ribosome itself. Some AFs catalyze RNA cleavage (endo- and exoRNases) or have roles in RNA modification (snoRNPs and base methyltransferases), RNP remodeling (helicases, ATPases and GTPases) or protein modification (kinases, phosphatases, SUMO conjugases, etc.). Other AFs were recently suggested to test subunit functionality and to act as placeholders that mask important ribosomal sites until subunit maturation is achieved(Fig. 3a).


Figure 3: ‘Placeholders’ and molecular mimicry in biogenesis of ribosomes and snoRNPs. 
(a) During ribosomal-subunit biogenesis, placeholders mask important functional sites to prevent premature activity. On the small ribosomal subunit precursor (pre-40S), depicted with rRNA and RPs in gray, AFs
(in bright colors) are masking the decoding sites (DCS) at the base of helix (h) 44, as well as tRNA- and mRNA-binding sites (localization of functional sites in Box 1). The position of RPS14 discussed in b is indicated in the incipient platform (Pt). The inset shows that the DCS is distorted in the precursor subunit and acquires its functional configuration only after AF displacement. Adapted with permission from ref. 13, AAAS. 
(b) Molecular mimicry in ribosome biogenesis. In mature 40S ribosomal subunits, the ribosomal protein RPS14 interacts with three 18S rRNA helices (h23, h24 and h45; bottom). During biogenesis, the rRNA-binding surface of RPS14 is blocked by the adenylate kinase FAP7 to prevent premature integration of RPS14 into the 40S subunit (top). The crystal structure of FAP7–RPS14 was solved in archaea and the RPS14–18S rRNA interaction in budding yeast46 (PDB 4CVN, 3U5B and 3U5C). 
(c) Molecular mimicry in snoRNP assembly. During H/ACA snoRNP assembly, the AF SHQ1 blocks the snoRNA-binding surface of the pseudouridine synthase CBF5 (top), occupying the position of the guide RNA in mature snoRNPs (bottom). The crystal structure of the CBF5–SHQ1 complex was solved in budding yeast42 and the CBF5-guide interaction in Archaea93 (PDB 3HAY and 3ZV0). L7ae (archaeal NHP2) and NOP10 are core H/ACA proteins.

By design, their displacement is a prerequisite for catalytic activation of the ribosome. Yet, in the absence of known motifs in their protein sequences, the functions of most AFs remain unknown, and further structural work must be conducted to understand precisely what they do. The description of human pre-rRNA processing has lagged far behind that of budding yeast, partly because of the assumption that processing is evolutionarily conserved. However, 625 human nucleolar proteins were recently tested for functions in ribosome biogenesis; of those, 286 were shown to be required for rRNA processing, including 74 without yeast counterparts. Forty percent of these 286 new processing factors were linked to human diseases, mostly cancers and genetic disorders. Nearly one-third of the human factors identified perform additional or distinct processing functions as compared to those of their yeast homologs. Typically, factors involved in small-subunit processing in yeast are also required for large-subunit maturation in humans and vice versa. For example, the exosome subunit Rrp6 is required for the 3′-end formation of 5.8S rRNA in yeast, and its human homolog EXOSC10 is also needed for 18S rRNA 3′-end maturation. These differences could reflect higher coordination between the machineries involved in the processing of the small and large subunits in humans compared to yeast. In fast-growing yeast cells, up to 70% of nascent pre-rRNAs are cleaved cotranscriptionally within ITS1. This is not known to occur in vertebrates, at least to this extent, thus probably offering additional opportunities for interactions between early- and late-acting processing machineries. The evolutionary trend in ribosome biogenesis is toward increased complexity. A remarkable example is that sequences equivalent to 5.8S and 28S are collinear in bacteria and archaea but are separated by ITS2 in eukaryotes. Across eukaryotes, variable expansions in mature rRNAs occur more often, and there is a greater number and size of noncoding spacers, additional cleavage sites, alternative pathways and new unique AFs as described above. The trend is matched by a 25-fold-higher complexity of the human nucleolar proteome compared to that of yeast, and it correlates with the divergence of a single fibrillar compartment into two morphologically separate nucleolar layers: the fibrillar centers and dense fibrillar components.

pre-ribosomal RNA modification, processing and folding by snoRNAs
Ribosome biogenesis depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. snoRNAs are small, abundant, stable RNAs. So far, they have been found in all eukaryotes, and equivalents, known as small RNAs (sRNAs), are present in Archaea. snoRNAs act in pre-rRNA modification, processing and folding through Watson-Crick base-pairing with their substrates. There are three classes of snoRNAs: box C/D, box H/ACA and MRP, all of which are active as snoRNPs, in intimate association with core proteins. Assembly of the snoRNPs themselves requires dozens of AFs. 

My comment: Imagine that you find an assembly site of large complex machine, a 3D printer. There you find nearby, around that 3D printer, smaller machines, that are employed in the making of other machines. And these other machines are employed to make elementary components of that 3D printer, which is employed to make  3D printers identical of itself, but it can also make a myriad of other machines depending on the algorithmic assembly instructions that he is fed with. In other words, machines are assisting in the making of other machines, which are employed in the making of subunits of other machines.... amazing !! 

We make a machine for a specific purposes by first designing it, then drawing up a list of components that will be needed, then acquiring the components, and then building the machine. For that, we need a plan. A goal. We need a view of the finished system. We need to know in advance which pieces will be relevant. We need to know beforehand the key use of the elementary components. It is the whole machine that makes sense of its components. The whole is more than the sum of the parts. Subsystems are highly INTERLOCKED within the universal system The interlocking is tight and critical. At the center everything depends on everything. Such a multiple interlocking of functions made through machines that are made by other machines could only have been a product of intelligent design.

Such a 3D printer requiring such complex assembly can never be instantiated by evolution or stochastic random chance. Those have no plan nor foresight, and no view of the finished system and its function. Mechanisms without foresight cannot  know in advance which pieces might be relevant. It cannot know how to make the machines, that make the machines, that make the small parts of the 3D printer, nor how to assemble the whole thing. It is the whole machine that makes sense of its components and assembly manufacturing. The whole is presupposed by all the parts. At the center everything depends on everything. Natural selection or chance would not select the parts to fabricate a machine, which has no use by its own, unless there is foresight and foreknowledge of its use to achieve the distant overarching design goal.

Ribosomes are 3D printers of proteins. Human ribosomes require over 200 Small nucleolar RNAs (snoRNAs) which are a class of small RNA molecules that primarily guide chemical modifications of  ribosomal RNAs for their biogenesis. And the assembly of the snoRNPs themselves requires dozens of Assembly Factors (proteins with a wide range of biochemical activities). In other words, assembly factors are assisting in the assembly of Small nucleolar RNAs, which are themselves involved in the assembly of ribosomalRNA's, which are themselves elementary components necessary to build the large integrated macromolecular ribosome translation systems.

Most box C/D and box H/ACA snoRNPs drive RNA modification24 (Box 2). 



The conserved boxes are bound by proteins important for snoRNA stability, nucleolar targeting and snoRNP function. Box C/D and H/ACA snoRNAs, ranging in size from 60 to 200 nt and 120 to 250 nt, respectively, are associated with four core proteins, including the enzymes that mediate rRNA modification. For box C/D snoRNPs, this is the methyltransferase Fibrillarin (FBL; NOP1 in yeast), and for box H/ACA, this is the pseudouridine synthase Dyskerin (DKC1 (also known as NAP57); CBF5 in yeast). Other snoRNPs are involved in pre-rRNA processing. Among those, the RNase MRP is in a class of its own. Composed of the MRP RNA (268 nt in humans, 340 nt in yeast) bound by ten core proteins, MRP is involved in pre-rRNA processing at site A3 in ITS1 in yeast, a function that is apparently not conserved in humans. MRP shares eight proteins with RNase P, which is active in tRNA 5′-end maturation, and their RNAs are structurally related. Specialized members of both C/D and H/ACA families are also involved in RNA processing in yeast and vertebrates. Of those, the box C/D snoRNA U3, also referred to as the ‘SSU-processome’, takes part in small-subunit maturation in yeast and humans, whereas U8 is required for large-subunit processing in vertebrates. Finally, snoRNAs probably have largely underestimated roles in pre-rRNA folding because they act through extensive Watson-Crick base-pairing and can have multiple targets on pre-rRNAs, located far apart from each other. This is the case for U3, involved in eukaryotic central-pseudoknot formation, and U8, acting during vertebrate ITS2 maturation. The central pseudoknot is a universally conserved long-range interaction within the 18S rRNA that has a crucial role in the overall folding of the small subunit. Both U3 and U8 interact with pre-rRNAs during biogenesis, sequestering complementary sequences and thereby preventing their premature interaction. In Escherichia coli, which lacks U3, the timing of central-pseudoknot formation is also regulated through establishment of alternate base-pairing, but that is accomplished by cis-acting elements within the pre-rRNA itself. This is also true of ITS2 maturation in yeast, which lacks U8. The strategies used for central-pseudoknot formation and ITS2 maturation provide two remarkable cases of increased complexity in rRNA processing.

Parallels between biogenesis of ribosomes and Small nucleolar RNAs (snoRNPs) 
During ribosome assembly, structural reorganization of RNAs and core proteins occurs, and this is accompanied by a reduction of RNP complexity through the successive and regulated loss of associated AFs. This process triggers catalytic activation of the ribosome (Fig. 3a). Thus it seems that, having passed the initial assembly stages, ribosome subunit biogenesis pathways essentially become ‘disassembly’ pathways (examples in refs. 33–35). The assembly of snoRNPs shares many similarities with ribosome assembly. Though they are less complex than ribosomes, dozens of AFs are involved in snoRNP production. For instance, as in ribosome biogenesis, small proteinaceous subcomplexes of AFs are sequentially recruited to nascent precursor RNPs; subsequently, there is a progressive loss of associated factors, and there are checkpoints or ‘delays’ regulating catalytic activation.
The AFs involved in snoRNP biogenesis comprise both class-specific and shared AFs. For example, NAF1 and SHQ1 are specific to H/ACA RNP assembly, whereas Hsp90 and its cochaperone R2TP trigger structural remodeling during synthesis of both C/D and H/ACA RNPs36. Furthermore, there is abundant cross-talk in the assembly of distinct RNPs, which sometimes rely on the same AFs. Small Cajal body RNPs (scaRNPs) are structurally related to snoRNPs, but they localize in Cajal bodies and participate in the modification of spliceosomal small nuclear RNAs37. SHQ1, involved in H/ACA snoRNPs assembly, is also required for the synthesis of H/ACA scaRNPs and the mammalian H/ACA telomerase RNP. In addition,15.5K (Snu13), the primary box C/D binder, initiates the recruitment of specific core proteins to box C/D snoRNPs, box C/D scaRNPs and spliceosomal U4 snRNP. Several ribosome and snoRNP AFs use the principle of ‘molecular mimicry’, reproducing specific protein-RNA interactions through protein-protein contacts. During ribosome assembly, the adenylate kinase FAP7 interacts with ribosomal protein RPS14 by mimicking its contacts with the rRNA, thus regulating the timing of RPS14 integration in 40S (Fig. 3b). During H/ACA snoRNP assembly, the AF SHQ1 interacts with the pseudouridine synthase CBF5 across the RNA-binding interface, occupying the position of the guide RNA in mature snoRNPs. This precludes interaction of CBF5 with the snoRNA until the snoRNP has adequately matured and prevents premature RNA modification (Fig. 3c). Other AFs ‘mask’ core protein–binding sites on the snoRNA, further regulating the timing of snoRNP assembly. NAF1, a structural homolog of the H/ACA core protein GAR1, has been suggested to act as a ‘placeholder’ until it is replaced by GAR1 for final snoRNP maturation and catalytic activation. Similarly, NUFIP (yeast Rsa1) has been suggested to hold core proteins together in immature particles and to act as an adaptor between 15.5K-bound RNP precursors and Hsp90–R2TP, which binds to 15.5K (Snu13) in a manner predicted to exclude interactions occurring in mature snoRNPs.



a) Tandem repeats occur in DNA when a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other.[

1. https://pediaa.com/what-cellular-structure-is-responsible-for-manufacturing-ribosomes/
2. https://tel.archives-ouvertes.fr/tel-01878354/document
3. https://www.sciencedirect.com/science/article/pii/S0014579314003007
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600399/
5. https://www.nature.com/articles/nrc.2017.104
6. https://sci-hub.ren/10.1038/nrc.2017.104
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551681/
8. https://www.wikigenes.org/e/gene/e/851191.html
9. https://sci-hub.ren/10.1038/nsmb.2939
10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555582/
11. https://www.bzh.db-engine.de/group/56/martin%20koš/setLang=en

The process of making a single ribosome is a herculean task, and is one of the most metabolically expensive activities of a cell. 1 In vigorously growing yeast cells, it requires the activity of all three RNA polymerases, accounting for 70% of total transcription, 90% of pre-mRNA splicing and more than 25% of translation. 

Saccharomyces cerevisiae is a species of yeast (single-celled fungus microorganisms). The S. cerevisiae genome is composed of about 12,156,677 base pairs and 6,275 genes, compactly organized on 16 chromosomes.

In Saccharomyces cerevisiae, nearly 7000 nucleotides of pre-rRNA must be accurately transcribed, cleaved, folded, chemically modified by 71 snoRNPs directing either 2′-O-methylation or pseudouridylation, and assembled with 78 ribosomal proteins (r-proteins) to form one mature ribosome. Despite the immensity of this task, about 2000 new ribosomes are produced each minute in yeast (~7500 subunits per minute in HeLa cells), leading to the presence of ~200 000 ribosomes in each cell (~10 million in each HeLa cell)

A number of large- and small-scale studies have identified sub-complexes involved in ribosome biogenesis.  There are numerous enzymes involved in maturation steps of pre-ribosomes.  Eukaryotic cells contain large populations of small nucleolar RNA-protein complexes, called snoRNPs, and these complexes mediate the formation of modified nucleotides in ribosomal RNA (rRNA) and facilitate cleavage of rRNA precursors. They contain a component called snoRNA. Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs.

Dozens of Assembly Factors are involved in snoRNP production

snoRNAs: Biogenesis, Structure and Function  2
The snoRNAs function in association with specific proteins and thus form ribonucleoproteins (snoRNPs). To date, about 80 different snoRNAs have been found in the yeast Saccharomyces cerevisiae, and almost twice as many in human cells. Some snoRNAs are much more abundant than others, for instance there is about 200 000 copies of U3 in human cells. The snoRNAs are relatively short molecules ranging between 60 and 600 nucleotides, but most of them are in the range of 70–200 nucleotides. They can be grouped into two major families called C/D and H/ACA; this classification is based on conserved sequence motifs (Figure below).


Structure of guide snoRNAs and mode of action. 
The schematic secondary structure of snoRNAs are presented on the left; the blue line represents the target RNA that gets modified. The conserved sequences are boxed and indicated in colour. Models for the selection of specific position to be modified are shown on the right. In (a), box C/D snoRNAs are characterized by conserved motifs C and D(green) that form a kink-turn (K-turn) or alternate boxes C’ and D’ (orange) that could also form a K-turn; 2’-O-ribose methylation is performed on the rRNA residue that is base-paired to the fifth position upstream from box D (or D’). In (b), H/ACA snoRNAs adopt a hairpin-hinge-hairpin-tail structure where box H (red) is found in the hinge region and box ACA (red) is found three nucleotides upstream of the 3’- end; each hairpin usually contains an internal loop called pseudouridylation pocket where C formation in rRNA occurs on the first unpaired U residue upstream from box H or ACA (usually at a distance of 14 to 16 nucleotides from the box).

Role of snoRNAs in Pre-rRNA Processing and Modification
The small- and large-subunit rRNAs are transcribed as part of a large precursor that is subsequently cleaved and trimmed to release mature rRNA products, the 18S rRNA, which is part of the small subunit (SSU), and the 5.8S and 25–28S rRNAs, which are found in the large subunit (LSU) of the ribosome. In contrast to pre-mRNAs, which begin to be processed (spliced) before completion of synthesis, pre-rRNAs are generally fully transcribed before cleavages occur. Post-transcriptional modifications are made on the precursors, not the mature products, and some modifications could be prerequisites to processing events (Figure 2).


Figure 2 Pre-rRNA modification and processing. 
The large pre-rRNAs encode the small ribosomal subunit rRNA (18S) and the large ribosomal subunit rRNAs (5.8S and 25–28S). The coding sequences are separated by spacer sequences named 5’ETS (5’ external transcribed spacer), ITS1 (the internal transcribed spacer 1), ITS2 (internal transcribed spacer 2), and 3’ETS (3’ external transcribed spacer). The guide snoRNPs are involved in modification reactions; C/D snoRNPs direct 2’-O-ribose methylation (Me), and H/ACA snoRNPs direct pseudouridylation (C). The processing snoRNPs (represented by spheres) are involved in cleavage reactions. Cleavages are likely orchestrated by a large complex containing many snoRNPs, which is referred to as the ‘processome’ (by analogy with the spliceosome). The transcribed spacers are removed by a series of endo- and exonucleolytic reactions, and mature rRNAs are liberated and packaged into ribosomal subunits.

snoRNA-associated Proteins
snoRNA and its associated proteins can be quite complex. U3 snoRNA and all of its associated proteins for example are very large and complex, containing over 30 different proteins, and it has been coined the SSU processome.  The snoRNAs function in the form of ribonucleoproteins (RNPs).

Expression of snoRNAs
Rapid and efficient production of large quantities of sno-RNAs may be crucial for cell survival. Cajal bodies (CBs) are spherical nuclear organelles that are often seen juxtaposed or near the nucleolus. CBs appear to be sites of biogenesis or recycling of various ribonucleoproteins (RNPs)

My comment: So these RNP's have an own factory, where they are synthesized. That indicates their importance. Quite remarkable. So the factory (Cajal bodies) is annexed and near another factory (nucleolus), and both are involved in the making of ribosomes, which are protein factories. Interlinked factories, that make factories, hosted inside a giant factory, the cell, which makes other factories ( cells ).  

These organelles harbour a specific subset of small RNAs called scaRNAs (small Cajal body-specific RNAs) that are structurally and functionally indistinguishable from snoRNAs. Most scaRNAs are implicated in posttranscriptional
modifications of polII-transcribed spliceosomal snRNAs (U1, U2,U4 and U5). snoRNAs assemble or mature in CBs before transiting to the nucleolus.

My comment: So this is the picture:Small nucleolar RNAs (snoRNAs) are involved in the Pre-ribosomal RNA Processing and Modification. And scaRNAs (small Cajal body-specific RNAs) are implicated in post-transcriptional modifications of snRNAs. This is analogously of machines (snoRNAs) making machines (snoRNAs) , that are employed in the processing of of subunits of other machines (ribosomes) which are themselves involved in making machines ( proteins, and subunits of ribosomes ). That is a catch22 situation. It takes ribosomes to make ribosomes. What came first ?

Biosynthesis, assembly, and transport of sno/scaRNPs 3
The Cajal body associated RNAs (sno/scaRNAs) follow a unique biosynthetic pathway before they are transported to the Cajal bodies. Both C/D and H/ACA sno/scaRNAs are synthesized in the nucleoplasm, processed, assembled with their respective proteins, and transported to the Cajal body

My comment: This is truly remarkable. For some reasons, these RNA's are made in the nucleoplasm, and not in the Cajal body. Transporting them to the Caja body means more complexity. 


A model of the biosynthesis and assembly of box C/D and box H/ACA RNPs is represented in this Figure.

Biosynthesis of scaRNAs
Nearly all sno/scaRNAs are intronic sequences that are freed from the primary transcript by endonucleases or by splicing after mRNA processing. Protein binding near sno/scaRNA terminals trigger exonucleases to degrade both ends of the intronic sequence until reaching the sno/scaRNA structure, where further degradation is inhibited by a bound protein and the mature sno/scaRNA is released

Assembly of scaRNPs
Binding of the 15.5K protein initiates the assembly of box C/D RNPs. In archaea, the L7Ae (15.5K in eukaryotes) and sno/scaRNA complex is recognized by nucleolar protein 5 (Nop5) (Nop56 and Nop58 in eukaryotes). Binding of L7Ae forms and stabilizes the K-turn which allows Nop5 and fibrillarin to join the complex. In eukaryotes, Nop58 binds first with fibrillarin and Nop56 joins the complex later. The N-terminal domain (NTD) is responsible for interaction with fibrillarin. scaRNAs are composite C/D and H/ACA box snoRNA. Assembly of H/ACA RNPs requires the specific chaperone, Shq1 which binds dyskerin (Cbf5 in archaea and NAP57 in rodents) to prevent degradation, aggregation, and binding to the premature RNA before co-transcriptional association. SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) 4

My comment: This adds another layer of complexity.  Pre-ribosomal RNA Processing and Modification requires. Small nucleolar RNAs (snoRNAs), which, in order to become functional, depend on post-transcriptional modifications of scaRNAs (small Cajal body-specific RNAs), which require for their assembly the specific chaperone, Shq1. In other words: A functional Ribosome requires subunits, which require for their processing  (snoRNAs), which require for their maturation (snoRNAs), which require for their assembly specific chaperone, Shq1


Crystal structure of the Shq1-specific domain (SSD).
(A) Domain organization of Shq1. The CS domain and SSD are labelled. 
(B) Ribbon representation of the SSD structure in cross-eye stereo-view. The secondary structural elements are indicated. The central helix α8 is green, the outer helical circle is blue, the inner helical circle is orange and the C-terminal structural elements are red. The N- and C-termini are marked by ‘N' and ‘C'. Dots are used to represent the disordered loops. 
(C) Topology diagram of the SSD structure. 
(D) Sequence alignment of the SSD. Ninety Shq1 proteins were aligned, but only the S. cerevisiae (Sc) and H. sapiens (Hs) sequences are shown. Residues that are conserved in at least 95, 80 and 60% of the 90 Shq1 proteins are shaded by black, grey and light grey, respectively. The secondary structure elements observed in the free SSD structure are indicated above the alignment. Residues with solvent accessible surface buried by at least 30 and 10 Å2 owing to Cbf5 association are marked by red solid and open circles, respectively. 5

snoRNAs are highly conserved throughout evolution. They are found in mammals, amphibians, fishes, plants, yeast, trypanosomes and even archaebacteria. Another fascinating feature of the snoRNA world is the presence of brain-specific snoRNAs in mice and humans. The very large number of snoRNAs, the diversity of their structure and biological roles (modification of rRNAs, tRNAs, mRNAs and snRNAs) in addition to the fact that they are highly conserved throughout evolution may reflect that they are life essential, and evidence of intelligent design. 


https://en.wikipedia.org/wiki/Small_Cajal_body_specific_RNA_13


https://en.wikipedia.org/wiki/Small_Cajal_body_specific_RNA_18




Eukaryotic ribosome biogenesis at a glance 2013
https://jcs.biologists.org/content/126/21/4815
Ribosomes are fundamental macromolecular machines that function at the heart of the translation machinery, allowing the conversion of information encoded within mRNA into proteins. The 80S ribosome (named for its apparent sedimentation velocity) is a ribonucleoprotein complex that comprises two ribosomal subunits, a large 60S subunit [containing the 25S, 5.8S and 5S rRNA, and 46 ribosomal proteins (r-proteins)] and a small 40S subunit (containing the 18S rRNA and 33 r-proteins).


rRNA synthesis and processing as a source of ribosome diversity
Being at the core of ribosome function, rRNAs are under tremendous selective pressure. Therefore, their sequence and secondary and tertiary structures are extremely well conserved, although some variation occurs in the expansion segments. Expansions are generally located far from the functional core of the ribosome and therefore are liable to only subtly influence translation. However, sequence diversity at the level of mature rRNAs has been described, involving either regulated expression of specific rDNA genes, activation of cryptic processing or simply nucleotide polymorphism. In addition, cases of constitutive differential processing occur in all eukaryotes, such as the short and long forms of 5.8S rRNA. In Plasmodium, different rDNA genes are expressed at specific developmental stages, and alternate forms of rRNAs, with sequence heterogeneity in the variable expansions, are incorporated into ribosomes that cannot substitute for function in yeast cells. In the naked mole-rat, ribosomes undergo constitutive clipping in the 28S rRNA to result in the excision of a 263-nt fragment from a variable region. Interestingly, clipped mole-rat ribosomes are substantially more accurate than unclipped mouse ribosomes49. At multiple places in eukaryotic rRNA-processing pathways, alternative routes can be followed, and there are cases in which cleavages that normally occur concomitantly are uncoupled, thus leading to production of new rRNA intermediates (for example, production of 43S and 26S) (Fig. 2). This presumably affects the kinetics of other facets of ribosome biogenesis, such as rRNA modification. This may be why aggressive human breast cancer cells accumulate elevated levels of 43S intermediates and are characterized by altered rRNA modification profiles that make them less prone to translate internal ribosome entry site (IRES)-dependent mRNAs51.

Role of RNA modifications and snoRNAs.
The mature rRNAs are extensively covalently modified at >100 sites. The most numerous rRNA modifications are 2’-O-ribose methylation and pseudouridylation, which are guided by snoRNAs. In addition, many nucleotides in rRNAs are modified by specialized enzymes. While it is well accepted that the modifications contribute to the rRNA conformation and ribosome function, the roles of individual or clusters of modifications is not fully understood. Significantly, mutations in modifying enzymes have been linked to human diseases, e.g. dyskeratosis congenita or the Bowen-Conradi syndrome. Furthermore, a lack of certain modifications was shown to extend life-span of multiple organisms.11

Specialized ribosomes – formation and function? Role in ageing?
In the recent years it has become clear that ribosomes with differently modified rRNA or diverse protein composition coexist in cells. It has been proposed that these distinct ribosomes might be specialized for translation of a subset of mRNAs.



Let us suppose someone would provide you with blueprints/assembly and manufacturing instructions to make a 3D printer. It would contain all the detailed and precise specifications  to make each single elementary part and subunit, a list of the raw materials, and all instructions to assemble and integrate the subunits. You would also receive all raw materials like plastic, glass, metal, resins, carbon fibers, graphene, nitinol etc. Now you would hand over both, the materials, and the instructional blueprints, to a highly trained and specialized team of engineers, each with its specialization, and asking them to manufacture the 3D printer. You would give them any time they would ask for. What they would lack, is the facility (factory) with the manufacturing equipment (which requires an entirely different set of specifications and expertise) that produce the subunits of the 3D printers and the production lines to assemble them. But this manufacturing equipment,  machines/robots, and production lines on the other hand, require as well precise specifications and factories in order to be made. In other words, what is required, are factories, that make factories, that make the subunits, and assembly of the 3D printer. The more automated and autonomous the process would have to be, the more complexity would have to be added. On top of that, the entire assembly process would have to be without external input of information, nor intelligent action. Everything would have to be preprogrammed, and if something during the manufacturing process breaks down, there would have to be mechanisms to detect the errors, and repair them. The entire manufacturing process would also have to be self-regulated, and under constant assembly control. Also the environment of the factories would have to be constantly monitored, and any change, like temperature, pH, air purity etc. detected and fixed.  It is evident, that the entire process requires foresight.  Things with specific purposes are always first the product of a creative mind, and then physically instantiated. With the end in mind, a blueprint is made which permits the implementation of the desired result. What i described, is what analogously happens in the biogenesis process of the Ribosome. 

Ribosomes are large multimolecular machines that synthesize proteins from amino acids in living cells.The process of making a single eukaryotic ribosome is a herculean task. Ribosome biogenesis is an essential major metabolic process in all organisms. The making of ribosomes in eukaryotes requires the coordinated action of all three RNA polymerases, numerous small nucleolar RNAs (snoRNAs) and several hundred protein factors. The highly dynamic and complex pathway of ribosome synthesis is directly or indirectly linked to various celullar processes. The Tor signaling pathway controls ribosome biogenesis at different levels. Ribosome biogenesis is a complex, dynamic process involving the coordinated transcription, processing, modification, and structural remodeling of immature ribosomal RNA (rRNA) and binding of ribosomal proteins.  In eukaryotes, ribosome assembly spans three cellular compartments, beginning in the nucleolus and continuing in the nucleoplasm, with final stages of maturation occurring in the cytoplasm. To ensure efficient and accurate construction of ribosomes, eukaryotic ribosome assembly is facilitated by several hundred protein assembly factors (AFs), which include nucleases, RNA helicases, nucleoside triphosphatases, and scaffolding proteins, among others. 11 Ribosome assembly is hierarchical, with primary binding r-proteins participating in the formation of binding sites for later-entering r-proteins. Large ribosomal subunit assembly occurs in blockwise parallel pathways. 

In Saccharomyces cerevisiae (single-celled fungus microorganisms), nearly 7000 nucleotides of pre-rRNA must be accurately transcribed, processed and assembled with 78 ribosomal proteins (r-proteins) to form one mature ribosome. Despite the immensity of this task, about 2000 new ribosomes are produced each minute in yeast (~7500 subunits per minute in human HeLa cells), leading to the presence of ~200 000 ribosomes in each cell (~10 million in each human HeLa cells) Several sub-complexes are involved in ribosome biogenesis.  There are numerous enzymes involved in maturation steps of pre-ribosomes.  Eukaryotic cells contain large populations of small nucleolar RNA-protein complexes, called snoRNPs, and these complexes mediate the formation of modified nucleotides in ribosomal RNA (rRNA). They contain a component called snoRNA. Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs  , transfer RNAs   and small nuclear RNAs  .

Consider snoRNAs as machines that make the subunits of a more complex machine ( the ribosome ) Dozens of Assembly Factors are involved in snoRNP production. So these are OTHER machines that make snoRNAs  machines.  The snoRNAs function in association with specific proteins and thus form ribonucleoproteins (snoRNPs).  snoRNA and its associated proteins can be quite complex. U3 snoRNA and all of its associated proteins for example are very large and complex, containing over 30 different proteins, and it has been coined the SSU processome. 


The making of snoRNAs: Rapid and efficient production of large quantities of sno-RNAs is crucial for cell survival. Cajal bodies (CBs) are spherical nuclear organelles that are often seen juxtaposed or near the nucleolus. CBs appear to be sites of biogenesis or recycling of various ribonucleoproteins (RNPs) 

My comment: So these RNP's have an own factory, where they are synthesized. That indicates their importance. Quite remarkable. So the factory (Cajal bodies) is annexed and near another factory (nucleolus), and both are involved in the making of ribosomes, which are protein factories. Interlinked factories, that make factories, hosted inside a giant factory, the cell, which makes other factories ( cells ).  

These organelles harbour a specific subset of small RNAs called scaRNAs (small Cajal body-specific RNAs) that are structurally and functionally indistinguishable from snoRNAs. Most scaRNAs are implicated in posttranscriptional
modifications of polII-transcribed spliceosomal snRNAs (U1, U2,U4 and U5). snoRNAs assemble or mature in CBs before transiting to the nucleolus.

My comment: So this is the picture:Small nucleolar RNAs (snoRNAs) are involved in the Pre-ribosomal RNA Processing and Modification. And scaRNAs (small Cajal body-specific RNAs) are implicated in post-transcriptional modifications of snRNAs. This is analogously of machines (snoRNAs) making machines (snoRNAs) , that are employed in the processing of of subunits of other machines (ribosomes) which are themselves involved in making machines ( proteins, and subunits of ribosomes ). That is a catch22 situation. It takes ribosomes to make ribosomes. What came first ?

The sno/scaRNAs follow a unique biosynthetic pathway before they are transported to the Cajal bodies. Both C/D and H/ACA sno/scaRNAs are synthesized in the nucleoplasm, processed, assembled with their respective proteins, and transported to the Cajal body

My comment: This is truly remarkable. For some reasons, these RNA's are made in the nucleoplasm, and not in the Cajal body. Transporting them to the Caja body means more complexity. 

Biosynthesis of scaRNAs:  Protein binding near sno/scaRNA terminals trigger exonucleases to degrade both ends of the intronic sequence until reaching the sno/scaRNA structure, where further degradation is inhibited by a bound protein and the mature sno/scaRNA is released

Assembly of scaRNPs:  Binding of the 15.5K protein initiates the assembly of box C/D RNPs. scaRNAs are composite C/D and H/ACA box snoRNA. Assembly of H/ACA RNPs requires the specific chaperone, Shq1 to prevent degradation, aggregation, and binding to the premature RNA before co-transcriptional association. SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) 4

My comment: This adds another layer of complexity.  Pre-ribosomal RNA Processing and Modification requires. Small nucleolar RNAs (snoRNAs), which, in order to become functional, depend on post-transcriptional modifications of scaRNAs (small Cajal body-specific RNAs), which require for their assembly the specific chaperone, Shq1. In other words: A functional Ribosome requires subunits, which require for their processing  (snoRNAs), which require for their maturation (snoRNAs), which require for their assembly specific chaperone, Shq1

snoRNAs are highly conserved ( they have not changed). They are found in mammals, amphibians, fishes, plants, yeast, trypanosomes and even archaebacteria. Another fascinating feature of the snoRNA world is the presence of brain-specific snoRNAs in mice and humans. The very large number of snoRNAs, the diversity of their structure and biological roles (modification of rRNAs, tRNAs, mRNAs and snRNAs) in addition to the fact that they are highly conserved throughout evolution may reflect that they are life essential, and evidence of intelligent design.

My comment: Above shows why George Church was right. He asked: If I were to be an intelligent design defender, that's what I would focus on; how did the ribosome come to be? Things with specific purposes are always first the product of a creative mind, and then physically instantiated. With the end in mind, a blueprint is made which permits the implementation of the desired result. DNA, the blueprint of life, instantiates the making of the ribosome, its operation and  processes. Therefore, the ribosome, maybe the most central player in biochemical processes, the factory of proteins, is the product of Gods mind.

Assembly and structure of the SSU processome — a nucleolar precursor of the small ribosomal subunit April 2018 6
The small subunit processome is the first precursor of the small eukaryotic ribosomal subunit. During its assembly in the nucleolus, many ribosome biogenesis factors, an RNA chaperone, and ribosomal proteins associate with the nascent pre-rRNA. Biochemical studies have elucidated the rRNAsubdomain dependent recruitment of these factors during SSU processome assembly and have been complemented by structural studies of the assembled particle. Ribosome biogenesis factors encapsulate and guide subdomains of preribosomal RNA in distinct compartments. This prevents uncoordinated maturation and enables processing of regions not accessible in the mature subunit. By sequentially reducing conformational freedom, flexible proteins facilitate the incorporation of dynamic subcomplexes into a globular particle. Large rearrangements within the SSU processome are required for compaction into the mature small ribosomal subunit. 

A particle containing the entire 35S pre-rRNA is termed the 90S pre-ribosome. A 23S pre-rRNA species (containing the 50 ETS, 18S rRNA and ITS1 cut at site A3) has been associated with the SSU processome. This intermediate was also found to accumulate as a result of mutations and depletions of small subunit ribosomal proteins, ribosome assembly factors or components of the exosome. More recently, the accumulation of the 23S pre-rRNA species was also observed in response to cellular stress, nutrient starvation or as a result of mTOR inhibition. Here we discuss how advances in our biochemical understanding of many SSU processome factors combined with recent cryo-EM reconstructions have facilitated a mechanistic understanding of the assembly and structure of this pre-ribosomal particle.

Early events in small subunit assembly
SSU processome assembly proceeds in a chronological and co-transcriptional manner (Figure 1). 


Figure 1 Co-transcriptional assembly of the small subunit processome. 
Schematic representation of early events of small subunit assembly in the nucleolus and subsequent maturation resulting in the mature cytoplasmic SSU. A section of the rDNA locus is shown with 50 ETS and ITS1 colored in yellow and 18S colored in red (50 domain), green (central domain) and slate (30 domain). Factors visualized in the assembled SSU processome are shown as schematic outline while transient components are listed and colored according to the rRNA domain they are associated with. The SSU processome and its major components are shown in detail below.

The 50 external transcribed spacer (50 ETS), which precedes the 18S rRNA, enables the recruitment of several protein factors and the U3 snoRNP and leads to the formation of an early assembly intermediate termed the 50 ETS particle. Eukaryotic ribosome biogenesis is initiated by the sevensubunit UtpA complex (Utp4, Utp5, Utp8, Utp9, Utp10, Utp15 & Utp17). This initially flexible complex was shown to specifically interact with the first half of the 50 ETS by in vivo pull-down assays as well as UV-cross- linking and analysis of cDNA (CRAC).UtpA binding and the presence of additional parts of the 50 ETS appear to be required for the subsequent recruitment of the six-subunit UtpB complex (Utp1/ Pwp2, Utp6, Utp12, Utp13, Utp18 & Utp21) and the U3 snoRNP (U3 snoRNA, Nop1/fibrillarin, Snu13, Nop56 & Nop58) (Figure 1). UtpB chaperones an RNA duplex formed by the 50 ETS and U3 snoRNA and later parts of the 30 domain of the 18S rRNA. U3 snoRNA acts as a vital RNA chaperone during the ensuing assembly steps where it base-pairs with two regions of the 50 ETS and two regions of the 18S rRNA. This prevents the pre-mature formation of the central pseudoknot — a tertiary RNA structure that determines the relative positions of rRNA domains in the mature small subunit. U3 snoRNA dictates the topology of the growing particle by first base-pairing with the 50 ETS through its 30-hinge and 50-hinge and then with the 18S rRNA through its Box A (18S nucleotides) and Box A0 (18S nucleotides 1111–1122). As transcription continues, these base-pairing interactions presumably result in a reduction of conformational freedom of preribosomal RNA, thereby directing RNA folding and the topology of the entire particle.

With the completion of the 50 ETS, the Mpp10 complex (Imp3, Imp4 & Mpp10), individual assembly factors (Bud21, Fcf2, Utp7, Utp11, Sas10 & Sof1) and later the A1 site nuclease Utp24, join UtpA, UtpB and U3 snoRNP to finalize the 50 ETS particle (Figure 1). With the exception of Utp7, Sof1, and Utp24, these factors lack canonical protein folds and act as multi-functional binding partners, which may, similar to U3 snoRNA, reduce conformational freedom by interacting with multiple other proteins as the assembly of the small subunit processome continues. Conceptually, these peptides and U3 snoRNA could therefore allow an initially flexible
array of subdomains to be stabilized in a co-transcriptional manner to result in a rigidified SSU processome.

The 50 ETS particle is an architectural scaffold onto which individual subdomains of the small ribosomal subunit (50, central, 30 major and 30 minor domains) can be assembled. The purification of affinity-tagged truncated rRNA mimics has shown that each domain is associated with a distinct set of ribosome assembly factors (Figure 1). However, many of these proteins and RNAs such as snR10 and snR30 , perform transient roles during the assembly of the SSU processome and are not part of the stable particles recently studied by cryo-EM . Beyond the initial assembly events, flexible regions of Sas10, Utp18 and later Lcp5 contain exosome-interacting motifs, which may facilitate processing, degradation or recycling of SSU processomes .

The core of the SSU processome is composed of the 50 ETS at the base of the structure, ribosomal RNA on top and U3 snoRNA, which reaches from the outside into the center, where all interactions with the 50 ETS and 18S rRNA occur (Figure 2a).


Figure 2Structural organization of the yeast small subunit processome.
(a) RNA molecules of the SSU processome are shown as surfaces with 50 ETS (yellow), U3 snoRNA (red) and pre-18S (light-grey). Structural elements of RNAs and helices of the 50 ETS are indicated. (b) Ribosomal proteins
are represented in dark-grey, non-ribosomal assembly factors in transparent light-blue, and RNA species as in (a). (c) Surface representation of centrally located ribosome assembly factors. (d) Visualization of the complexes UtpA (blue), UtpB (red), U3 snoRNP (purple), UtpC (light-blue), the Nop14-Noc4 complex (brown) and the Mpp10 complex (orange). (e) Surface representation of all individual components of the small subunit processome.

The tips of the 5' ETS helices project freely into the solvent. This explains the length variation of this pre-ribosomal spacer which contains under 600 nucleotides in C. thermophilum, 700 nucleotides in yeast and more than 3600 nucleotides in humans. U3 snoRNA not only rigidifies the structure of the 50 ETS through its 50 and 30 hinges, but further outlines the positions of the 5', central and 3' major domains by providing spatial constraints of the regions that base-pair with the 5' end of the 18S rRNA (Box A) and a region between the central and 30 domains (Box A0). Fifteen ribosomal proteins are predominantly adopting the same conformations and binding sites as in the mature ribosome (Figure 2b). Only rpS6, rpS18 and rpS23 assume slightly different, yet near-mature conformations . A large shell of more than 51 ribosome assembly factors encapsulates pre-ribosomal RNA and ribosomal proteins. The innermost layer of this shell is formed by extended peptide-like proteins, which weave through the entire particle (Figure 2c). Members of this group include the multi-modular proteins Faf1, Lcp5, Mpp10, Sas10, Fcf2, Rrt14, Utp11 and Utp14, which are characterized by their unusual folds and many interaction partners as described later. Several large multi-subunit complexes (Figure 2d) as well as individual ribosome assembly factors (Figure 2e) provide the outer shell of the SSU processome. In agreement with previous functional data, UtpA stabilizes the first half of the 50 ETS and is located at the bottom of the particle. Through multiple interactions with UtpA and the 5' ETS, UtpB components Utp18 and Utp6 likely make the first contact before the other four subunits of the UtpB complex are fully integrated into the SSU processome . Interestingly, while both UtpA and UtpB share a common evolutionary origin as four subunits of each complex form a structurally related tetrameric arrangement with their C-terminal domains, their roles within the SSU processome are different. UtpA serves as a foundation of the particle to initiate its assembly, while UtpB provides a binding platform for the 3' hinge, and stabilizes spatially distant parts of the assembly including RNA elements near the 3' end of the 18S rRNA. The UtpC complex is positioned at the top of the particle, where it interacts with the tip of helix 44. The outermost shell of the SSU processome is formed by many additional ribosome assembly factors (Figure 2e). These include the acetyltransferase/helicase Kre33, which rests on the Bms1-Rcl1 GTPase complex at the top of the structure, and the methyltransferase Emg1, which is positioned on a lateral extension formed by the Nop14/Noc4 complex. Lastly, Utp20, Utp10, Rrp5 and the Nop14/Noc4 complex provide large helical repeat structures to support and bridge distant regions of the particle.

The role of flexible proteins with multiple conserved binding motifs
The SSU processome is a eukaryote-specific particle with an intricate network of elongated peptides containing multiple binding motifs (Figure 3a). Strikingly, these long linkers are used to bridge conserved protein–protein
interaction motifs (Figure 3b–e).

Figure 3: Peptides connect distant sites within the SSU processome via conserved binding motifs. 
(a) Schematic protein–protein interaction diagram of selected SSU processome components represented as spheres or lines with interacting elements as helices or strands. The Utps (U-three proteins) are labeled with their respective number. (b–e) Detailed views of Utp18 and Utp14 
(b), Mpp10 
(c), Bms1 
(d) and the U3 snoRNP 
(e) with proteins shown as surface or cartoon, colored according to conservation with residues conserved more than 90% highlighted as spheres. Direct interaction partners are depicted as surface or a grey dashed line. All proteins are colored by conservation from lighter to darker shades. Clustal was used to align manually curated sequences (H. sapiens, S. cerevisiae, G. gallus, D. melanogaster, S. pombe, C. elegans, D. rerio, A. thaliana, A. gambiae, P. troglodytes, R. norvegicus, M. musculus, B. taurus, S. scrofa) and plotted onto the structure using Homolmapper.

Utp18 contains an N-terminal extension, which interacts with parts of the UtpB complex (Utp21 & Utp6) as well as the UtpA complex (Utp10), the U3 snoRNP (Nop58) and the exosome-associated helicase Mtr4 via the arch-inter-acting-motif (AIM) (Figure 3b). These peptides enable initial flexibility within the yeast UtpB complex followed by a later stable association with the SSU processome . 

Similar peptide-like inter-actions are employed by Utp14, which interacts with three globular folds of other SSU processome components (Utp7, Sof1, Utp6) to stabilize nucleotides around the A1 cleavage site . Additionally, Utp14 recruits Dhr1, the helicase required to unwind the U3 snoRNA (Figure 3b). Mpp10 fulfills many different roles within the SSU processome (Figure 3c). Chemical cross-linking and biochemical studies have shown that its flexible N-terminus interacts with rpS5 and Sas10. In addition, the structured C-terminus of Mpp10 is involved in stabilizing Bms1 (Figure 3d), bridging between Imp3, Imp4 and UtpB and supporting a remodeled pre-rRNA segment near helix 44. The U3 snoRNP is a central nexus for several peptide-like motifs (Figure 3e). Here the C-terminal halves of Utp11, Sas10 and Fcf2 bind to conserved surfaces of Nop1 while their N-terminal halves together with an Mpp10 segment are used to stabilize Bms1 upon its incorporation within the SSU processome (Figure 3d). Sas10 also employs molecular mimicry by occupying the same position as rpS30 in the mature small ribosomal subunit (Figure 3d and e).

The observed dynamics of SSU processome assembly together with a complete high-resolution structure of this particle now allow us to propose a three-dimensional assembly model in S. cerevisiae (Figure 4a).

Figure 4 Three-dimensional maturation model of the Saccharomyces cerevisiae small-subunit processome. 
(a) Co-transcriptional assembly of the SSU processome as a function of transcription of rRNA regions (280 nucleotides of 50 ETS, 50 ETS, 50 domain, central domain and 30 domain). 50 ETS (yellow), rRNA domains (white) and complexes such as UtpA (light-blue), UtpB (red), U3 snoRNP (purple, red), the Mpp10 complex (orange) and additional proteins, are shown as surfaces. Bound peptides are shown as cartoon with their initially flexible tails as dashed lines. As maturation progresses, these tails recruit additional factors and become ordered. The pre-rRNA shown in the intermediates is schematically indicated below each particle and colored in darker shades. As this model is based on the mature SSU processome structure, regions that are presumed to be flexible in earlier states are highlighted accordingly. (b, c) Comparative locations of rRNA domains within the SSU processome [PDB 5wlc] 
(b) and the mature small ribosomal subunit [pdb 4v88] 
(c). Individual rRNA domains are colored identically with 50 domain (blue), central domain (red), 30 domain (green) and shown as spheres superimposed onto transparent outlines of the particles. In the SSU processome, the flexible helix 44 is indicated as schematic outline. Rearrangements of rRNA domains from the SSU processome (b) that are necessary to obtain the positions within the mature small ribosomal subunit 
(c) are indicated with arrows. The central U3 snoRNA Box A and Box A0 are colored in purple. RNA elements disordered in the SSU processome are indicated in lighter shades in the mature SSU.

Conceptually this model takes into account the roles of initially flexible peptide sequences (Utp11, Bud21, Sas10 and Mpp10) and RNA sequences (U3 snoRNA 50 and 30 hinges as well as Box A and Box A0), which co-transcriptionally dictate the locations of rRNA and protein folds and reduce conformational freedom to stabilize the maturing SSU processome (Figure 4a). Major enzymatic and structural changes are required to
move beyond the SSU processome towards the mature small ribosomal subunit (Figure 4b). Enzymatic steps include the unwinding of the Box A and Box A0 duplexes by RNA helicases such as Dhr1 [46], cleavage
at site A1 by the nuclease Utp24 [24,25,48], and the replacement of the GTPase Bms1 by the structurally related factor Tsr1. In addition, structural changes such as rotational and translational movements of the
central and 30 domains with respect to the 50 domain are required (Figure 4b). The formation of the central pseudoknot and its surrounding elements, the formation of inter-domain base-pairing interactions between
the central and 50 domains and the incorporation of additional ribosomal proteins are further steps requir-ing control during later stages of the assembly pathway.

Perspectives
During the last five years, our understanding of the small subunit processome has progressed from a list of protein factors to an initial assembly model and a near atomic description of its structure. However, the functional integration of this giant particle in the cellular context is still poorly understood. Little is known about the specific signaling that results in either the arrest of ribosome assembly or its resumption. While the SSU processome has been identified as a storage particle, the mechanisms leading to its accumulation or subsequent processing remain unclear. Furthermore, structural inter- mediates that capture earlier conformations or released states of the SSU processome will be required to understand the mechanistic principles of this essential eukaryotic assembly intermediate.

Integrity of the P-site is probed during maturation of the 60S ribosomal subunit 2012 Jun 11 7
The complexity of ribosome structure would appear to present an extreme challenge to a cell to ensure the correct assembly and function of the ribosome. Because defects in assembly would likely lead to reduced function and fidelity of the ribosome, strategies ensure the proper function of newly assembled ribosomes. Maturation of the pre-60S subunit involves the recycling of export factors, the removal of placeholder proteins, and the assembly of several critical r-proteins. We have recently established the order of events of the cytoplasmic maturation pathway of the LSU. Two different ATPases carry out one series of protein exchanges, leading to the release of the export receptor Arx1. The ribosome stalk, which is critical for recruiting and activating translation factors, is assembled separately. These two series of events are prerequisite for the function of the GTPase Efl1, which together with Sdo1 releases the shuttling protein Tif6. Tif6 binds to the intersubunit bridge B6, making contacts with the sarcin-ricin loop (SRL), Rpl23, and Rpl24, thereby blocking 40S joining. Efl1 is homologous to the translation elongation factor eEF2 (elongation factor G [EF-G] in prokaryotes, whereas Sdo1 is orthologous to the human Shwachman–Bodian–Diamond syndrome protein, mutations in which cause Shwachman–Bodian–Diamond syndrome, an autosomal recessive bone marrow failure disease. In the last known step, which depends on the prior release of Tif6, the export adaptor Nmd3 is released from the LSU by the GTPase Lsg1.

eEF2 and EF-G promote mRNA–tRNA translocation during translation. After peptidyl transfer, the peptidyl tRNA rapidly shifts to the hybrid A/P position through a natural ratchetlike motion of the subunits. During translocation, EF-G is recruited to the GTPase-associated center of the ribosome by the L7/L12 stalk. GTP hydrolysis by EF-G induces a conformational change in the protein that drives translocation of the peptidyl tRNA from the A/P position into the P/P position.

Cytoplasmic assembly of the P0/P1/P2 protein stalk (the eukaryotic equivalent of L10/L7/L12) is necessary for recruitment and activation of Efl1 to induce the release of Tif6. In this model, Efl1 utilizes the known function of the stalk to recruit and activate GTPases during translation for a biogenesis-specific function. A loop of the LSU protein Rpl10 is also intimately involved in the release of Tif6 from the LSU. This loop, which we will refer to as the P-site loop, extends toward the catalytic center of the ribosome, contacting the acceptor stem of the P-site tRNA. Mutations in this loop prevent the release of Tif6. Mutations in Efl1 bypass the effects of these P-site loop mutations. These Efl1 mutations are predicted to destabilize domain interfaces and facilitate conformational changes analogous to those that eEF2 undergoes during translocation. Our data suggest that in addition to interrogating the correct assembly of the stalk, Efl1 interrogates the P-site of the ribosome in a more rigorous assessment of the integrity of LSU assembly than previously recognized. The utilization of a translocation-like activity during biogenesis suggests that the newly assembled ribosomal subunit undergoes a test drive before being released into the active pool of ribosomes engaged in translating mRNAs.

The post-transcriptional steps of eukaryotic ribosome biogenesis 14 April 2008 8
One of the most important tasks of any cell is to synthesize ribosomes. In eukaryotes, this process occurs sequentially in the nucleolus, the nucleoplasm and the cytoplasm. It involves the transcription and processing of pre-ribosomal RNAs, their proper folding and assembly with ribosomal proteins and the transport of the resulting pre-ribosomal particles to the cytoplasm where final maturation events occur. In addition to the protein and RNA constituents of the mature cytoplasmic ribosomes, this intricate process requires the intervention of numerous protein and small RNA trans-acting factors. These transiently interact with pre-ribosomal particles at various stages of their maturation. Most of the constituents of preribosomal particles have probably now been identified and research in the field is starting to unravel the timing of their intervention and their precise mode of action. Moreover, quality control mechanisms are being discovered that monitor ribosome synthesis and degrade the RNA components of defective preribosomal particles. 

Tightly-orchestrated rearrangements govern catalytic center assembly of the ribosome 27 February 2019 9
The catalytic activity of the ribosome is mediated by RNA, yet proteins are essential for the function of the peptidyl transferase center (PTC). In eukaryotes, final assembly of the PTC occurs in the cytoplasm by insertion of the ribosomal protein Rpl10 (uL16). We determine structures of six intermediates in late nuclear and cytoplasmic maturation of the large subunit that reveal a tightly-choreographed sequence of protein and RNA rearrangements controlling the insertion of Rpl10. We also determine the structure of the biogenesis factor Yvh1 and show how it promotes assembly of the P stalk, a critical element for recruitment of GTPases that drive translation. Together, our structures provide a blueprint for final assembly of a functional ribosome.

Ribosomes are the molecular machines that all cells depend on for protein synthesis. Its two fundamental functions, decoding messenger RNAs and polypeptide synthesis, are separated into the small subunit and large subunits, respectively. Despite using RNA for catalysis, ribosomes are ribonucleoprotein particles, and proteins surrounding the peptidyl transferase center (PTC) are essential for function. In eukaryotes, the ribosomal subunits are largely preassembled in the nucleolus where the ribosomal RNAs are transcribed. However, ribosomal subunits are exported to the cytoplasm in a functionally inactive and immature state, requiring the further addition of ribosomal proteins and the removal of transacting factors that block ligand binding sites. As a consequence, the assembly of ribosomes is coupled to their nuclear export.

In budding yeast, nuclear export of nascent pre-60S subunits requires the 

export adapter Nmd3, 
the mRNA export factor Mex67-Mtr2, 
the degenerate methionyl amino peptidase Arx1, and 
several other proteins. 

However, only Nmd3 appears to have a universally conserved role as an export factor in eukaryotes. Interestingly, Nmd3 homologs are also found in archaea, suggesting that the protein has a function in ribosome assembly that predates the evolution of the nuclear envelope and its role as an export factor. Nmd3 is a multidomain protein that we and others previously showed spans the entire joining face of the 60S subunit. Its eIF5A domain occupies the E site, while additional domains bind in the P site and occlude the A site, rendering the joining face inaccessible to transfer RNAs and other large subunit ligands. A small entourage of additional biogenesis factors accompanies the pre-60S to the cytoplasm. Among these factors, Tif6 blocks association with the small subunit to prevent premature engagement of the assembling 60S.

In the cytoplasm, the pre-60S particle follows a hierarchical pathway of assembly events coordinated with the release of biogenesis factors. Cytoplasmic maturation is initiated by the AAA-ATPase Drg1, which is recruited to the subunit and activated via Rlp2422, a paralog of the ribosomal protein Rpl24. Release of Rlp24 appears to be coordinated with the release of the GTPase Nog123, which disrupts the A site while its C-terminal extension is inserted into the polypeptide exit tunnel. Downstream completion of the subunit requires assembly of the P (L7/L12) stalk, which recruits and activates the GTPases of the translation cycle25, and insertion of Rpl10 (uL16), to complete the PTC. Molecular genetics analyses showed that assembly of the P stalk requires the dual-specificity phosphatase Yvh1 to release the placeholder protein Mrt4, a paralog of the P stalk protein P0 (uL10). Similarly, functional interactions among RPL10, NMD3 and LSG1, encoding a second GTPase, suggest an interplay among these factors in promoting the loading of Rpl10 and the release of Nmd3. The insertion of Rpl10 into the pre-60S particle completes the subunit, priming it for quality control. The integrity of the PTC is subsequently assessed in a test drive which uses molecular mimics of translation factors to license the subunit for translation by the release of Tif6. Defects in this test drive are associated with Shwachman–Diamond syndrome in humans. While extensive molecular genetics and biochemical studies over the past 20 years have provided a framework for understanding the complex process of cytoplasmic maturation, the mechanisms for assembly of both the P stalk and the PTC have remained elusive.

Recent advances in cryo-electron microscopy (cryo-EM) give us the ability to resolve intermediates of ribosome assembly at near atomic resolution. Multiple structures of nucleolar and nuclear pre-60S intermediates have been reported. However, only a single high-resolution structure of a native cytoplasmic intermediate has been reported. Here, we have used cryo-EM to determine the structures of a series of intermediates of 60S maturation that reveal the dynamic changes in RNA conformations and protein exchanges required for assembly of the P stalk and completion of the PTC.



1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035417/
2. https://sci-hub.ren/10.1038/npg.els.0003813
3. https://sci-hub.ren/10.1016/j.tcm.2017.08.002
4. http://genesdev.cshlp.org/content/25/22/2398
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242979/
6. https://sci-hub.ren/10.1016/j.sbi.2018.01.008
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373404/
8. https://link.springer.com/article/10.1007/s00018-008-8027-0
9. https://www.nature.com/articles/s41467-019-08880-0

.

Nervous-Like Circuits in the Ribosome

In the past few decades, studies on translation have converged towards the metaphor of a “ribosome nanomachine”;. Many studies have shown that to perform an accurate protein synthesis in a fluctuating cellular environment, ribosomes sense, transfer information, and even make decisions. This complex “behavior” that goes far beyond the skills of a simple mechanical machine has suggested that the ribosomal protein networks could play a role equivalent to nervous circuits at a molecular scale to enable information transfer and processing during translation. We analyze here the significance of this analogy and establish a preliminary link between two fields: ribosome structure-function studies and the analysis of information processing systems. This cross-disciplinary analysis opens new perspectives about the mechanisms of information transfer and processing in ribosomes and may provide new conceptual frameworks for the understanding of the behaviors of unicellular organisms.

Proteins may constitute computational elements in cells. In unicellular organisms, protein-based circuits act in place of a nervous system to control the behavior. Because of the high degree of interconnection, systems of interacting proteins act as neural networks trained to respond appropriately to patterns of extracellular stimuli.  The recent analysis of r-protein networks in the ribosomes of the three kingdoms updates and further enhances this intriguing hypothesis. R-protein networks form complex circuits that differ from most known protein networks, in that they remain physically interconnected. These networks display some features of communication networks and an intriguing functional analogy with sensory-motor circuits found in simple organisms. These networks may play at a molecular scale, a role analogous to a sensory-motor nervous system, to assist and synchronize protein biosynthesis during translation. However, we must be aware that the nerve circuits do not have exactly the same properties that the ribosomal proteins circuits have, even in simple organisms having the most primitive nervous systems that are known.

Many of the ribosome functional properties go far beyond the skills of a simple mechanical machine.

Many experimental studies have shown that—as well summarized in the title of paper of Rhodin and Dinman—“an extensive network of information flow” through the ribosome during protein biosynthesis. First, growing evidence indicates that ribosome functional sites (RNA binding sites, decoding centre, peptidyl transferase centre (PTC), peptide exit tunnel) continually exchange and integrate information during the various steps of translation. For example, an allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation. In addition, the overall ribosome dynamics are modulated by the three tRNA site occupancies and their aminoacylation status. Additionally, several studies have demonstrated long-range signaling between the decoding centre that monitors the correct geometry of the codon-anticodon and other distant sites such as the Sarcin Ricin Loop (SRL) or the E-tRNA site [16,17]. The peptidyl transferase centre (PTC), the large-subunit rRNA active site where peptide bond formation is catalyzed, is also a key node of allosteric communication. PTC and the A-site communicate and are coordinated through the universal protein uL3. Moreover, specific r-proteins of the ribosomal tunnel play an active role in the translation regulation or co-translational folding by sensing the nature of the nascent peptide and communicating to the PTC or the exit sites. In addition, recent studies have extended the scope of ribosome sensing systems to a higher level in describing the molecular mechanisms of a quality sensor of collided ribosomes in eukaryotes and showed that sensing may also involve higher-order ribosome architectures to monitor the translation status.

Ribosome Choreography during Protein Biosynthesis
Second, to perform the biosynthesis of proteins, the ribosome must synchronize extremely complex movements by combining small and large-scale motions such as the ratchet-like motion between the two subunits. More than 21 hinges have been identified within the rRNAs for accomplishing independent unit motions in bacterial ribosome. Could Brownian molecular motions be efficient and fast for a system with so many freedom degrees without control? Brownian motion is convincingly suggested to be at the ambient origin of the process driving the basic motions of the ribosome, then rotation and elongation can be thermally driven. It has also been suggested that an out of equilibrium stochastic process, a variant of Totally Asymmetric Exclusion Process (TASEP), can describe approximately the basic sequence of ribosome’s movements. However, this sequence is certainly of a more elaborate nature than a pure TASEP, needing the intervention of external factors for regulation and also an internal control and adaptation, probably helped by the rRNA, mRNA, tRNA and the r-proteins. Interestingly, remote communication processes have been recently shown to participate in the coordination of complex ribosomal movements during translation, thus suggesting that ribosomal motions may be helped by external synchronization systems.

How information transit between distant functional sites and how this information is integrated and processed to enable an optimized ribosome activity in a fluctuating cellular environment remains one of the major challenges in the research on the ribosome. Many studies have already addressed this question and revealed that long-range communication occurs in the ribosome and perform information transfer between remote functional sites. This allosteric communication has been shown to either involve rRNA or r-proteins. However, the vocabulary frequently used in these recent studies implicitly reveals the limits of the machine metaphor: “Sensing”, “communicating” and even “taking decisions” are indeed generally employed for describing autonomous organisms and are rarely used for a machine.

In view of the extraordinary ribosome properties, it could be asked today if the ribosome is a device, an “intelligent machine”, a “Turing machine” or just an organism instead of an organelle? The later view implies that the ribosome has autonomous “behaviour” in the cell and is fully adapted to perform translation in a fluctuating cellular context. This apparently naïve view has two hypothetical implications. First, it infers that the “ribosome behaviour” would be supported by the equivalent of a nervous system at a molecular scale. Second, in an evolutionary point of view, it would lead to generalizing the concept of a “primordial endosymbiosis” to an early prokaryotic cellular origin. This view fits well with recent papers that propose that the ribosome is a self-replicating intermediate between the RNA world and cellular life and may constitute a missing link in the evolution of life. These papers have shown indeed that the rRNA contains genetic information that encodes the self-replication machinery, all tRNAs and key ribosomal proteins. Thus, similar to the mitochondria endosymbiosis in eukaryotic cells, RNA/protein organisms like the ribosomes may have joined the cytoplasm of a primitive prokaryotic cell. Cells would have subsequently evolved to protect and optimize pre-existing functions.

My comment: How could ribosomes have joined the cytoplasm of a primitive prokaryotic cell, if prokaryotic cells need the very own ribosome in order to live? 

Our recent analysis of r-proteins networks formed by tiny but highly conserved contacts between r-protein extensions noticed an intriguing analogy of these networks with nervous circuits . This study proposed that in addition to the previously well-described allosteric mechanisms, r-protein networks could also contribute to transfer and process the “information flow” during translation. This provided an integrated framework that could be based, at least in part, on graph theory, to investigate information transfer and processing in the whole ribosome. 

D. Bray explicitly suggested the “neuron-protein equivalence” in protein circuits. Following Bray’s metaphor, r-proteins may be functionally comparable to neurons and their networks should, therefore, form nervous-like circuits able to transfer and process information at a molecular scale. We examined here this hypothesis knowing that if a protein behaves like a neuron at a molecular scale, it should have properties similar to a neuron: sensing, transferring and integrating information. R-protein networks would be also expected to display some similarities with an interacting ensemble of nervous circuits systems. We explore these analogies to evaluate their significance and their heuristic power in the understanding of the ribosome’s behaviour.

In fact, the metaphor is perhaps more appropriate and precise with the substructures of a central nervous system CNS, for instance, a collection of neuronal areas. This is indicated in particular by eukaryotic ribosomes: although there is a multiplication by ten of the number of bases in rRNA between C. elegans and primates and a corresponding growing complexity of the elements of the proteins, there are no noticeable changes of their r-protein networks. In comparison, the graph of the neuronal networks becomes more and more complex, the numbers of neurons and of their mutual connexions explode. However, in vertebrates, for instance, the whole plan of the CNS is relatively stable. Thus, a better analogy seems to occur between the r-proteins network and interconnected areas of the nervous central system (CNS). The neuronal assemblies become more complex as the individual proteins and their contacts do.

Many studies have already addressed this question and revealed that long-range communication occurs in the ribosome and perform information transfer between remote functional sites. This allosteric communication has been shown to either involve rRNA or r-proteins. However, the vocabulary frequently used in these recent studies implicitly reveals the limits of the machine metaphor: “Sensing”, “communicating” and even “taking decisions”

In the ribosome, the structural and functional organizations of the individual r-proteins suggest a close analogy with neurons in both the structural and functional points of view. Most ribosomal proteins are composed of a globular domain that is located at the surface of the ribosomal subunits and long filamentous extensions that penetrate deeply into the rRNA core. Some r-proteins are devoted to forming tiny interactions with substrates or products within the ribosome functional sites. A current view is that these interactions contribute to stabilizing the mRNA or tRNA substrates in their correct positions.

Sensing the functional sites to monitor the presence, the sequence or the correct orientation of the substrate may be, therefore, generalized to all the ribosomal functional sites.

My comment: In order to monitor the system, the ribosome must have previous knowledge of what should be, compare to what is, and if the error is detected, be able to act accordingly. This requires foresight and knowledge. 

It has been previously noted that the r-proteins “innervate” all the functional sites in a manner similar to a nervous system. The functions of sensing and stabilizing the substrates are not incompatible and may have co-evolved gradually for optimizing ribosome accuracy. 

My comment: First of all, the origin of the ribosome is a problem of abiogenesis, not evolution. Secondly, how could the ribosome work properly if accuracy was not established right from the get go, with all 11 different error check-and repair mechanisms in place? 

Many structural features and the electrostatic nature of the interactions suggest that common electrostatic mechanisms enable them to sense the different actors of translation through transient tiny interactions. The ribosome actively participates with the correct tRNA selection through the decoding centre. However, in contrast to polymerases, the recognition site that monitors the correct codon-anticodon pairing is about 70 Å away from the PTC where the polymerization takes place. The two sites must, therefore, communicate to perform accurate protein synthesis. 

My comment: Had this communication not to be fully set up and functional right from the start, otherwise, the right tRNA selection would not be established, and the entire system would break down and be non-functional? 

During the first step of decoding, the aminoacyl-tRNAs are delivered to the ribosome in a ternary complex with EF-Tu and GTP. The correct codon recognition induces a conformation change in EF-Tu and triggers the GTP hydrolysis leading to the dissociation of the EF-Tu-GDP complex from the ribosome, thus, freeing the aa-tRNA to accommodate into the PTC.  16S rRNA bases A1492, A1493, G530 and C1518 directly monitor the correct geometry of the codon-anticodon helix formed between the mRNA and the A-tRNA. It has been shown that these rRNA bases relay global structural changes of the small subunit that occur in response to the binding of cognate anticodon stem-loops in the decoding centre. This produces a “closed state” of the small subunit that, in turn, activates the EF-Tu GTP hydrolysis. Both biochemical and structural studies have demonstrated that uS12, uS3 and the interaction between uS4 and uS5 also play a key role in this process. r-proteins closely approach the decoding centre through electrostatic interactions involving conserved amino acids. Their side chains may, therefore, sense and transmit the local structural and electrostatic changes associated with the correct tRNA-mRNA pairing.

Molecular Synapses and Wires
In addition to sensing and/or stabilizing the tRNAs and the nascent peptides in the functional sites, r-protein extensions are systematically involved in r-protein contacts and form complex networks. Most of the r-protein interactions display well-defined interfaces that remain stable once the mature ribosome structure has been formed. With an average area of 200 A2, which is too tiny to be rationalized in terms of dimer stabilization, these highly phylogenetically conserved interfaces have play a specific role in inter-protein communication. Their analysis revealed that most of them share aromatic and basic residues involved in cation-π interactions that were interpreted as the “necessary minimum for communication”.

Molecular Communication
How the information is distributed in the network and between functional sites still remains to be determined. In the “classic” allostery, locally induced conformational changes are propagated to remote protein sites and modulate their properties. Ribosome allostery has been well documented. There are many communication rRNA and r-protein pathways between remote ribosome functional sites.   

One of the best analogies between ribosomal networks and simple neuronal networks is their modular structure. Even in simple organisms, as tunicates having a few hundred neurons, but being close to the vertebrates, several distinct sensori-systems work together. A similar modular organization is observed in the r-protein networks. Figure 9 shows that in the bacterial ribosome, r-proteins are organized into a few interconnected sub-networks grouped around the functional sites: decoding centre, tRNA sites, factor binding, PTC and tunnel. This r-protein network organization, therefore, suggests that the r-proteins not only sense the functional sites but also collectively share the information that circulates during translation. It is likely that the information about the occupancy/orientation of substrates in the different functional sites is locally integrated into the sub-networks and then transmitted and processed between the different sub-networks

Allostery: How Cells Do Remote Control
https://evolutionnews.org/2020/08/allostery-how-cells-do-remote-control/
And yet the high-fidelity cooperative action between the RNA and protein parts in the ribosome require allostery over vast distances (on the cellular scale). This is astonishing. How can they believe this level of remote control just happened?

The size and complexity of its structure and the diversity of covalent and noncovalent interactions required to execute 10 to 20 translation cycles per second with high fidelity demand the coordinated interactions of bases that span more than 390,000 Å of molecular surface. But how does the ribosome actually work? How is molecular information transferred between functional centers? What are the allosteric pathways and conduits of communication, and how do specific residues in the structure contribute to this communication? In this work, we apply a global coevolution method (SCA) to show that, despite differences in fundamental chemistry, RNA and protein appear to use a common logic of interaction and assembly.

Assembly! It’s not just the interaction to consider, but the assembly, too. The ribosome parts had to be translated first, and then the building blocks, consisting of two very different molecules (RNA and protein), have to be assembled to work. And that work involves remote controls, where “molecular information” is “transferred between functional centers” by means of “allosteric pathways and conduits of communication.” Those pathways, moreover, rely on “specific residues in the structure” that contribute to the communication. The authors are clearly in awe of what they are seeing. What does the talk about “co-evolution” contribute, other than fanciful storytelling? Let the “logic of interaction and assembly” speak for itself.




The possible communication pathways depicted by the blue, yellow and red arrays, between the functional sites within the r-protein network.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627100/

Nervous-Like Circuits in the Ribosome come to light

1. The set up of a language, and upon it, the programming of a completely autonomous communication network, which directs the operation of a complex factory, which during operation error checks and performs repairs, to make specific purposeful products, is always the product of an intelligent agency.

2. Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. They form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.

3. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confess that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: an intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.


Ribosomes have been called “intelligent machines”,  “Turing machines”, and "molecular factories". And a recent science paper, from 2019, has now even unraveled, that they contain nervous-like circuits. Ribosomes carefully sense, transfer, and process information, and amazingly, even make decisions. Ribosomal protein networks form complex circuits that differ from most known protein networks, in that they remain physically interconnected. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They have nodes of allosteric communication in a coordinated manner, remote communication processes, and information is integrated and processed to enable an optimized ribosome activity.  They, “Sense”, “communicate” and even “take decisions”. Isn't that awe-inspiring?  These networks are analogous with nervous circuits - the “neuron-protein equivalence” in protein circuits. They transfer and process information within itself at a molecular scale. These networks display some features of communication networks and an intriguing functional analogy with sensory-motor circuits found in simple organisms.  Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. Remarkably, they continually exchange and integrate information during the various steps of translation.

Through their superb integrated communication networks, ribosomes choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, which may also involve higher-order ribosome architectures, synchronize and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential. A recent Science paper: The last universal common ancestor between ancient Earth chemistry and the onset of genetics, published: August 16, 2018, informed that: LUCA had a ribosome and had the genetic code. This means the origin of the Ribosome cannot be explained by Darwinian evolution. 1  No wonder, does science confess, as in this paper: The Double Life of Ribosomal Proteins, for example: The origins and evolutionary history of these polypeptides remain an enigma. 2 But for us, theists, the enigma has an explanation: an intelligent cognitive agency, a powerful creator, God, during the creation week, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.  

More:
https://reasonandscience.catsboard.com/t1661-translation-through-ribosomes-amazing-nano-machines#7900

A recent science paper, from 2019, has given further light in regards of how Ribosomes perform their life-essential tasks, translating the information from messenger RNA to amino acids.  Ribosomes masterfully sense, transfer information, and, awe-inspiringly, even make decisions. This complex “behavior” that goes far beyond the skills of a simple mechanical machine has suggested that the ribosomal protein networks could play a role equivalent to nervous circuits at a molecular scale to enable information transfer and processing during translation. 

Proteins may constitute ingenious computational elements in cells. In unicellular organisms, protein-based circuits act strikingly in place of a nervous system to control the behavior. Because of the high degree of interconnection, systems of interacting proteins act as neural networks trained to respond appropriately to patterns of extracellular stimuli.  The recent analysis of ribosomal proteins (r-protein) networks in the ribosomes of the three kingdoms updates and further enhances this intriguing hypothesis. R-protein networks form complex circuits that differ from most known protein networks, in that they remain physically interconnected. These networks display some features of communication networks and an intriguing functional analogy with sensory-motor circuits found in simple organisms. These networks may play at a molecular scale, a role analogous to a sensory-motor nervous system, to assist and synchronize protein biosynthesis during translation. 

Many of the ribosome functional properties go far beyond the skills of a simple mechanical machine.

There is a superb extensive network of information flow” through the ribosome during protein biosynthesis. Ribosome functional sites (RNA binding sites, decoding centre, peptidyl transferase centre (PTC), peptide exit tunnel) continually exchange and integrate information during the various steps of translation. Long-range signaling between the decoding centre that monitors the correct geometry of the codon-anticodon and other distant sites.  The peptidyl transferase centre (PTC), the large-subunit rRNA active site where peptide bond formation is catalyzed, is also a key node of allosteric communication. In addition, recent studies have extended the scope of ribosome sensing systems to a higher level in describing the molecular mechanisms of a quality sensor of collided ribosomes in eukaryotes and showed that sensing may also involve higher-order ribosome architectures to monitor the translation status.

Ribosome Choreography during Protein Biosynthesis
To perform the biosynthesis of proteins, the ribosome must synchronize extremely complex movements with exquisite precision, by combining small and large-scale motions such as the ratchet-like motion between the two subunits. More than 21 hinges have been identified within the rRNAs for accomplishing independent unit motions in bacterial ribosome. Could Brownian molecular motions be efficient and fast for a system with so many freedom degrees without control?  Interestingly, remote communication processes have been recently shown to participate in the coordination of complex ribosomal movements during translation, thus suggesting that ribosomal motions may be helped by external synchronization systems.

In view of the extraordinary ribosome properties, it could be asked today if the ribosome is a device, an “intelligent machine”, a “Turing machine” or just an organism instead of an organelle? The later view implies that the ribosome has autonomous “behavior” in the cell. This apparently naïve view has two hypothetical implications. First, it infers that the “ribosome behavior” would be supported by the equivalent of a nervous system at a molecular scale. Second, in an evolutionary point of view, it would lead to generalizing the concept of a “primordial endosymbiosis” to an early prokaryotic cellular origin. This view fits well with recent papers that propose that the ribosome is a self-replicating intermediate between the RNA world and cellular life and may constitute a missing link in the evolution of life. These papers have shown indeed that the rRNA contains genetic information that encodes the self-replication machinery, all tRNAs, and key ribosomal proteins. Thus, similar to the mitochondria endosymbiosis in eukaryotic cells, RNA/protein organisms like the ribosomes may have joined the cytoplasm of a primitive prokaryotic cell. Cells would have subsequently evolved to protect and optimize pre-existing functions.

My comment: How could ribosomes have joined the cytoplasm of a primitive prokaryotic cell, if prokaryotic cells need the very own ribosome in order to live? 

Our recent analysis of r-proteins networks formed by tiny but highly conserved contacts between r-protein extensions noticed an intriguing analogy of these networks with nervous circuits . This study proposed that in addition to the previously well-described allosteric mechanisms, r-protein networks could also contribute to transfer and process the “information flow” during translation. This provided an integrated framework that could be based, at least in part, on graph theory, to investigate information transfer and processing in the whole ribosome. 

D. Bray explicitly suggested the “neuron-protein equivalence” in protein circuits. Following Bray’s metaphor, r-proteins may be functionally comparable to neurons and their networks should, therefore, form nervous-like circuits able to transfer and process information at a molecular scale. We examined here this hypothesis knowing that if a protein behaves like a neuron at a molecular scale, it should have properties similar to a neuron: sensing, transferring and integrating information. R-protein networks would be also expected to display some similarities with an interacting ensemble of nervous circuits systems. We explore these analogies to evaluate their significance and their heuristic power in the understanding of the ribosome’s behavior.

In fact, the metaphor is perhaps more appropriate and precise with the substructures of a central nervous system CNS, for instance, a collection of neuronal areas. This is indicated in particular by eukaryotic ribosomes: although there is a multiplication by ten of the number of bases in rRNA between C. elegans and primates and a corresponding growing complexity of the elements of the proteins, there are no noticeable changes of their r-protein networks.  Sensing the functional sites to monitor the presence, the sequence, or the correct orientation of the substrate may be, therefore, generalized to all the ribosomal functional sites.

My comment: In order to monitor the system, the ribosome must have previous knowledge of what should be, compare to what is, and if the error is detected, be able to act accordingly. This requires foresight and knowledge. 

It has been previously noted that the r-proteins “innervate” all the functional sites in a manner similar to a nervous system. The functions of sensing and stabilizing the substrates are not incompatible and may have co-evolved gradually for optimizing ribosome accuracy. 

My comment: First of all, the origin of the ribosome is a problem of abiogenesis, not evolution. Secondly, how could the ribosome work properly if accuracy was not established right from the get-go, with all 11 different error check-and repair mechanisms in place? 

Many structural features and the electrostatic nature of the interactions suggest that common electrostatic mechanisms enable them to sense the different actors of translation through transient tiny interactions. The ribosome actively participates with the correct tRNA selection through the decoding centre. However, in contrast to polymerases, the recognition site that monitors the correct codon-anticodon pairing is about 70 Å away from the PTC where the polymerization takes place. The two sites must, therefore, communicate to perform accurate protein synthesis. 

My comment: Had this communication not to be fully set up and functional right from the start, otherwise, the right tRNA selection would not be established, and the entire system would break down and be non-functional? 

Molecular Synapses and Wires
In addition to sensing and/or stabilizing the tRNAs and the nascent peptides in the functional sites, r-protein extensions are systematically involved in r-protein contacts and form complex networks. Most of the r-protein interactions display well-defined interfaces that remain stable once the mature ribosome structure has been formed. With an average area of 200 A2, which is too tiny to be rationalized in terms of dimer stabilization, these highly phylogenetically conserved interfaces have play a specific role in inter-protein communication. Their analysis revealed that most of them share aromatic and basic residues involved in cation-π interactions that were interpreted as the “necessary minimum for communication”.

Molecular Communication
How the information is distributed in the network and between functional sites still remains to be determined. In the “classic” allostery, locally induced conformational changes are propagated to remote protein sites and modulate their properties. Ribosome allostery has been well documented. There are many communication rRNA and r-protein pathways between remote ribosome functional sites.   

One of the best analogies between ribosomal networks and simple neuronal networks is their modular structure. Even in simple organisms, as tunicates having a few hundred neurons, but being close to the vertebrates, several distinct sensory-systems works together. A similar modular organization is observed in the r-protein networks. Figure 9 shows that in the bacterial ribosome, r-proteins are organized into a few interconnected sub-networks grouped around the functional sites: decoding center, tRNA sites, factor binding, PTC, and tunnel. This r-protein network organization, therefore, suggests that the r-proteins not only sense the functional sites but also collectively share the information that circulates during translation. It is likely that the information about the occupancy/orientation of substrates in the different functional sites is locally integrated into the sub-networks and then transmitted and processed between the different sub-networks.


Question: How did this awe-inspiring macromolecular nanomachine come to be? Could it acquire its amazing functionalities little by little, by trial and error. ?  

1. https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007518
2. https://www.cell.com/fulltext/S0092-8674(03)00804-3
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627100/

90S pre-ribosome transformation into the primordial 40S subunit 3

Production of small ribosomal subunits initially requires the formation of a 90S precursor followed by an enigmatic process of restructuring into the primordial pre-40S subunit. .

1. the remodeling RNA helicase Dhr1 engages the 90S pre-ribosome, followed by
2. Utp24 endonuclease–driven RNA cleavage at site A1, thereby
3. separating the 5′-external transcribed spacer (ETS) from 18S ribosomal RNA. Next,
4. the 5′-ETS and 90S assembly factors become dislodged, but this occurs sequentially, not en bloc.
5. Eventually, the primordial pre-40S emerges, still retaining some 90S factors including Dhr1, now ready to
6. unwind the final small nucleolar U3–18S RNA hybrid.

Our data shed light on the elusive 90S to pre-40S transition and clarify the principles of assembly and remodeling of large ribonucleoproteins. The formation of eukaryotic ribosomes requires transcription, processing, and modification of ribosomal RNA (rRNA) and the integration of ~80 ribosomal proteins. This highly complex process starts in the nucleolus with the transcription of a large rRNA precursor (35S in yeast, 47S in human), which contains the 18S rRNA of the 40S small subunit and the 5.8S and 25S rRNAs of the 60S large subunit, separated by the internal transcribed spacers ITS1 and ITS2 and flanked by the external transcribed spacers 5′-ETS and 3′-ETS. During transcription of the 35S pre-rRNA by RNA polymerase I, the earliest stable assembly intermediate, called the 90S pre-ribosome or small subunit processome, forms. This comprises more than 50 assembly factors, the U3 small nucleolar ribonucleoprotein (U3 snoRNP), the nascent pre-rRNA, and dozens of the small subunit ribosomal proteins. In the subsequent maturation steps, coordinated endonucleolytic cleavage at site A1 within the 5′-ETS and at A2 within the ITS1 is key to the separation of the 18S rRNA and 5.8S/25S rRNA (Fig. 1A), 


Fig. 1. Pre-rRNA processing and purification of 90S to pre-40S transition intermediates. 
(A) Sequence of rRNA processing events during 40S biogenesis in yeast with A0, A1, A2, A3, and D as cleavage sites on the pre-rRNA. 
(B) Analysis of splittag (Noc4-TAP–Dhr1-Flag) affinity-purified assembly intermediates by sucrose gradient centrifugation, followed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie staining or Northern blotting, using the RNA probes a, b, and c to detect the various indicated pre-rRNA forms (23S, 21S, 20S, 5′-ETS-A0). 
(C) SDS-PAGE of sucrose gradient fractions containing the pre-40S and 90S intermediates, with ribosome assembly factors identified by mass spectrometry indicated (bait proteins in red, factors shared by pre-40S and 90S in blue). See fig. S1B for the whole sucrose gradient.

which then follow independent biogenesis pathways. The remaining 5′-ETS is degraded by RNA nucleases, including the nuclear exosome. The four subdomains of the 18S rRNA (5′, central, 3′ major, and 3′ minor)  fold independently and to associate with their set of early ribosomal proteins of the small subunit (S-proteins) cotranscriptionally, before integrating into the 90S pre-ribosome. This integration happens in a reverse order, in which the 3′ major and 3′ minor domains incorporate first, followed by the central and 5′ domain. Together with its numerous associated 90S assembly factors, which predominantly are organized in modules (e.g., UTP-A, UTP-B, UTP-C, U3 snoRNP, Mpp10 complex, Bms1-Rcl1 complex, Kre33 module, Noc4 module, Krr1-Faf1 complex), the 90S preribosome is fully assembled and primed for subsequent A1 cleavage. After this cleavage, the 90S pre-ribosome is transformed into a primordial pre-40S intermediate (a process hereafter referred to as 90S transition) by a still-elusive mechanism; only later pre-40S intermediates have been described so far. The early 90S transition was initially uncovered by biochemical studies of Enp1, a ribosome assembly factor that is present in both early 90S and late pre-40S intermediates. However, many additional proteins have since been suggested to be intimately involved in this transition. Candidate factors include the methyltransferase Dim1, the KH domain proteins Krr1 and Pno1 (previously called Dim2), RNA helicase Dhr1, and the nuclear RNA exosome system. To decipher the mechanisms of this transition, we aimed at a structural analysis of 90S transition intermediates by cryo-EM.

 90S to pre-40S transition
The Noc4- Dhr1 and Dhr1-Dim1 have seven well-defined and distinct classes of pre-ribosomal particles, which were put in a temporal order covering the 90S transition (Fig. 2).


Figure 2: Cryo-EM analysis of 90S to pre-40S transition intermediates. 
CryoEM maps (top) and molecular models (middle) of distinct states of yeast 90S to pre-40S transition observed after split-tag affinity purification using Noc4-Dhr1 (all states) and Dhr1-Dim1 (states B2, Post-A1, Dis-A, Dis-B, Dis-C) are shown. Assembly factors and modules are labeled and compositional changes indicated. Bottom: Depiction of pre-18S rRNA density using 90S views with corresponding rRNA secondary structures; 40S views are shown in boxes (color code: magenta, 5′ domain; orange, central domain; cyan, 3′ major domain; green, 3′ minor domain).

These states are called B2, Pre-A1, Post-A1, and Dis-C. There are other three states :A, Dis-A, and Dis-B) permit rigid-body docking of molecular models (Fig. 2, figs. S2 to S6, and table S1). The first two particles in this series, states A and B2, closely resemble previously described 90S assembly intermediates, with a noncleaved A1 site as a characteristic feature (Fig. 2 and fig. S6). In the next state, Pre-A1, which is the last state preceding A1 processing, we observed the integration of helix 21 (h21) of the pre-18S rRNA at its mature position (Fig. 2 and fig. S6). Subsequently and concomitant with cleavage at site A1, major structural changes lead to the Post-A1 intermediate. Specifically, Pno1, together with h45 of the 18S rRNA, replaces the Krr1-Faf1 complex, bringing h45 from a peripheral to an integrated mature position. Furthermore, the remodeling helicase Dhr1 is observed for the first time in this post-A1 state intermediate, positioned at the site vacated by Pno1 (Fig. 2 and fig. S6). The succeeding group of intermediates comprises states Dis-A, Dis-B, and Dis-C (where Dis stands for dissociation), which represent the actual 90S to pre-40S transition intermediate states. Unexpectedly, the canonical pre-40S is not released as a whole entity after A1 cleavage by leaving a presumed 5′-ETS particle behind. Instead, several prominent 90S assembly factor modules dissociate one after the other, leading to a progressive simplification of these complexes (Fig. 2 and fig. S6). The last state of this series, Dis-C, after ultimately losing the conspicuous UTP-B module, resembles a 40S shape for the first time, clearly displaying the 5′, central (platform), and flexible and immature 3′ major and 3′ minor domains. Here, the characteristic h44 of the 3′ minor domain, which in preceding states is held in a defined immature position by UTP-B and UTP-C, is mostly delocalized. This is because the immature conformation is no longer stabilized by the 90S modules, and the mature position is still masked by the Bms1-Rcl1 complex (Fig. 2 and fig. S6). Conformational changes and mechanism of A1 cleavage In contrast to the expected en bloc release of a 5′-ETS particle upon A1 cleavage, we observed that the 5′-ETS RNA becomes increasingly disordered during a stepwise 90S transition. Helix H9 at the 3′ end of the 5′-ETS is the first to become detached from Utp20 (fig. S7, A to F). Next, after A1 cleavage, other prominent 5′-ETS helices (H3 to H8) disappear, leaving behind empty cavities in this part of the 90S intermediate (Fig. 3A and fig. S7, A to C). 


Fig. 3. Conformational, positional, and compositional changes upon A1 cleavage. 
(A and B) Dismantling of 5′-ETS RNA helices (yellow) from Pre-A1 (A) and Post-A1 (B) is shown with interacting U3 snoRNA (green) in transparent 90S density. The secondary structure of the 5′-ETS RNA is shown with observed helices in yellow and the dislodged helices in gray. (C and D) Volume representation of Utp20, the Kre33 module, and the 5′ and central domains of the 18S pre-RNA highlights the compaction from state Pre-A1 (C) to Post-A1 (D). Dashed lines indicate the movement of Utp20. (E and F) Model of the h1 region of the 18S pre-rRNA aligned at Utp24 shown before (E) and after (F) A1 cleavage. The catalytic center of Utp24 and the A1 cleavage site are indicated by dashed circles. (G) Differences in the 18S central domain region are highlighted in states Pre-A1 and Post-A1. (H) Ribbon representation of the platform region in state Post-A1. Nucleotides 1021 to 1025 of
the 18S rRNA are shown in magenta. (I) Model of Dim1 bound to its substrate adenosine 1782 of the pre-18S in state Post-A1. (J) Model of Pno1 in state Post-A1 with the 3′ end of the 18S pre-rRNA and interacting segments of Mpp10, Utp7, Utp14, and Utp21. (K and L) Models of yeast and human Pno1 bound to Utp14 (K) and NOB1 [(L), PDB ID 6G18], respectively, highlight overlapping interaction sites. Key residues are shown as sticks. (M) Ribbon representation showing the interaction of Dhr1 with Utp21 and Utp13 in state Post-A1. Amino acid abbreviations: F, Phe; I, Ile; K, Lys; L, Leu; M, Met; Q, Gln; R, Arg; S, Ser; T, Thr; W, Trp; Y, Tyr.


Only the first two helices, H1 and H2, and two distinct internal 5′-ETS segments that form short heteroduplexes with the 3′ hinge and 5′ hinge of U3 snoRNA remain in position (Fig. 3B and figs. S4 and S7C). The cause for the increased structural freedom or partial degradation of the 5′-ETS is not clear. Notably, despite the dismantling of the 5′-ETS RNA, the overall shape and composition of this 90S intermediate remained largely unchanged at first. 

During this 90S transition, a clear structural compaction was observed. Initially, in state B2, the C terminus of Utp20 exhibits a superhelical HEAT repeat structure that wraps around the rRNA expansion segment ES6c (fig. S7D). Then, after transition to the Pre-A1 state, the Utp20 superhelix adopts a more closed conformation (changing from a “C” to an “O” shape), caused by the movement of the immature 5′ domain toward the central domain. As a consequence, binding of Lcp5 and H9 to Utp20 is disrupted, allowing h21 (ES6d) to enter this previously occupied site (fig. S7, E and F). Similar to Utp20, the prominent Kre33 module also moves with the 5′ domain during this transition; however, the Kre33 module finally dissociates when the intermediate reaches the Post-A1 state (Fig. 3, C and D, and fig. S7, G to I). This brings the 5′ domain even closer to the central domain (fig. S7H), and when state Post-A1 is reached, the 5′ and central domains eventually interact and adopt a mature-like conformation (Fig. 3D and fig. S7I). This apparent domain compaction—together with the 5′-ETS RNA remodeling, which takes place during the first steps of 90S transition— results in a conformational state sufficiently dynamic to facilitate A1 processing: The state Pre-A1 shows the noncleaved A1 site ~50 Å away from the catalytic center of the PIN domain endonuclease Utp24, which was suggested to carry out this cut (Fig. 3E) (38–41). The Post-A1 state reveals the formation of a short RNA stemloop (h1) at the 5′ end of the mature 18S rRNA. The initial formation of this helix may have triggered endonucleolytic cleavage, because it results in repositioning the A1 site close to the catalytic center of the Utp24 endonuclease (Fig. 3F). Consistent with this interpretation, at the base of the newly formed h1 we can retrace theRNA sequence until the thirdnucleotide (uridine 3) of the cleaved and mature 5′ end of 18S rRNA. Such a sequence-independent but h1 stem-loop–dependent mechanism for A1 cleavage, requiring an arbitrary threenucleotide spacer relative to the h1 base, has been predicted by genetic studies from the Tollervey lab (42). Unexpectedly, A1 cleavage does not require unwinding of the Box A and Box A′ heteroduplexes formed by pre-18S rRNA and U3 snoRNA, the latter two being retained in the Post-A1 state (Fig. 3F). Together, the data show that A1 cleavage is initiated by remodeling and relocation of the pre-18S rRNA substrate rather than by movement of the endonuclease Utp24 toward the A1 cleavage site.

A1 cleavage coupled to Pno1 and 90S remodeling
A series of additional remodeling events can be observed when comparing the Pre-A1 and Post-A1 states, which includes the release of the Krr1-Faf1 complex and its replacement by the relocated Pno1 and h45 (Fig. 3, G and H, and fig. S8). With the translocation of Pno1 and the associated h45, the RNA platform region adopts a mature-like conformation, typically seen in both yeast and human later cytoplasmic pre-40S intermediates. This results in the formation of rRNA helices h19 and h25 (Fig. 3H), and, as a consequence, a new interface is formed and stabilized by the newly recruited methyltransferase Dim1 (Fig. 3I, fig. S9, and supplementary text), thereby rendering these rearrangements irreversible. The binding mode of Pno1 to h45 differs between the Pre-A1 and Post-A1 states (Fig. 3J and fig. S8B). Relocation of h45 relative to Pno1 is stabilized by the long C-terminal a helix of Utp7, which previously was bound to H7 of the 5′-ETS before its dismantling (Fig. 3J and fig. S8C). The interaction of Utp14 with Pno1 prevents premature recruitment of the endonuclease Nob1, which later catalyzes the last processing step of the pre-18S rRNA at site D (Fig. 3, K and L) (28). Finally, by relocating to the central platform, Pno1 and its clamped h45 dissociate from Utp21 and Utp13 of the UTP-B module (fig. S8B), allowing for the first time the recruitment of the RNA helicase Dhr1 to the 90S via a two-site contact to Utp21 and Utp13 (see below and Fig. 3M).

Successive factor shedding during 90S transition to primordial pre-40S
Following the formation of state Post-A1, the dismantled 5′-ETS allows for a cascade of structural and compositional changes that result in a stepwise reduction of the 90S complex (Fig. 4). 


Fig. 4. Successive shedding of factors during transition from 90S to primordial pre-40S. 
EM densities reveal the shedding process of major 90S assembly factor modules during transition from state Post-A1 to state Dis-C. Proteins, modules, and the U3 snoRNP are colored and labeled accordingly. The last remaining
helices of the 5′-ETS (H1, H2) and Dhr1 are shown in yellow. A local resolution-filtered map of Dhr1 was used in state Dis-C. The color-coded 18S pre-rRNA and the dismantled modules are shown below.

First, the Sof1 module (Utp7, Utp14, and Sof1) is released together with Utp6 from its binding site close to the former helix H9 at the 3′ region of the 5′-ETS, resulting in the state Dis-A. Then, in the second step, the UTP-A complex dissociates together with Utp18 and all protein components of the U3 snoRNP, leading to state Dis-B. In the last transition step from Dis-B to Dis-C, the core of the UTP-B complex and the remains of UTP-C are released. Also, the 5′-ETS, which served as RNA scaffold for all bindingmodules (UTP-A, UTP-B, UTP-C, and Imp3), is no longer detectable in this final intermediate. Of the U3 snoRNP, only a short segment of its RNA remains localized in states Dis-B and Dis-C, in particular the heteroduplexes with 18S rRNA; the rest becomes detached from these intermediates (Figs. 1B and 4). Interestingly, the domains of the pre-40S undergo rather limited maturation rearrangements during this shedding phase. Only rRNA expansion segments ES3a, ES3b, and ES6a to ES6d on the solvent side of the subunit adopt mature conformations, whereas the rest of the domains remain essentially unchanged (see boxes in Fig. 4 and fig. S10A).

Although this successive shedding of large protein modules is the distinct hallmark of the 90S transition, several structural changes occur in addition. First, Dhr1 moves from its previous UTP-B binding site (Fig. 3M) toward Box A in state Dis-C, thereby bringing the helicase into an ideal position to dismantle the last remaining U3::18S rRNA hybrid on the intermediate (fig. S10, B and C). Concomitantly, the Utp24 endonuclease becomes detectable
again in this state, where it occupies the binding site for the uS5 ribosomal protein. Moreover,Utp14, previously bound to Utp6 and helix H9 of the 5′ETS, relocates to a new position in state Dis-C, where it binds Pno1 and Dhr1 via four a helices (fig. S10, D to F). Notably, two arginine residues of Utp14 inhibit the endonuclease activity of Utp24 by coordinating active-site residues, thus allowing Utp24 to serve as an endonuclease-inactive placeholder (fig. S10, G to I). Sequential Dhr1 helicase function during the 90S to pre-40S transition Finally, the Dis-C intermediate sheds light on the diverse functioning of the Dhr1 helicase during the 90S transition by showing how it
is primed to remove the U3 snoRNA from its last contact point to the 18S rRNA, Box A. From state Post-A1 to state Dis-C, Dhr1 is tethered to the assembly intermediates via two invariant N-terminal helices: one that interacts with rRNA helix h13 at a site that is later occupied by Tsr1 (Fig. 5, A to C, and fig. S11, A and B), and another that binds to the methyltransferase Dim1 (Fig. 5D and fig. S9B). The catalytic C-terminal domain, however, binds distantly fromits N-terminal anchor region (Fig. 5, A and B, and fig. S11A). From state Post-A1 to state Dis-B, this globular part is bound to Utp13 and Utp21, which is mediated by a b barrel– like domain of Dhr1 (fig. S11C). In state Dis- C, however, after dissociation of Utp21, Dhr1 relocates to the Box A and h1 region, close to its U3 snoRNA substrate.Here, its b barrel–like domain is bound to the interface formed by Utp14, Pno1, and the 3′ region of the pre-18S rRNA (Fig. 5, E and F). The observed interplay between Utp14 and Dhr1 highlights the importance of Utp14 for the recruitment and relocation of Dhr1 during the 90S transition (36, 43–45). In our ensemble of 90S transition structures, Dhr1 was observed in two distinct conformations. First, between states Post-A1 and Dis-B, Dhr1 was in a conformation with an open RNA binding tunnel. Here, the N terminus of Pno1 prevents substrate binding and closing of the catalytic domain (Fig. 5G). This suggests that Dhr1 is bound to the 90S pre-ribosome in an adenosine diphosphate–bound, open conformation after A1 cleavage (fig. S11D). After the transition to state Dis-C and relocation of Dhr1 toward the Box A duplex, however, the helicase domain engages a segment of the U3 snoRNA as substrate (Fig. 5F). Furthermore, Dhr1 appears now in a closed conformation, resembling the adenosine triphosphate (ATP)–free form of previously reported RNA-bound DHX37, the human homolog of Dhr1 (Fig. 5H and fig. S11E) (18). We conclude from these data that after the transition from state Dis-B to state Dis-C, Dhr1 is in a closed apo-state, awaiting ATP binding, and through successive cycles of ATP hydrolysis could exert its pulling force to completely expel the U3 snoRNA from this primordial pre-40S intermediate.


How ribosomes are like Russian dolls

https://reasonandscience.catsboard.com/t1661-translation-through-ribosomes-amazing-nano-machines#8006

https://phys.org/news/2020-09-ribosomes-russian-dolls.html?fbclid=IwAR1nu4z70IXwlOloRrUTRLlkvNYulz1imSZoAADAAC0rPqP4M67w5dI0phM

Maturation ( biogenesis)  of the ribosome is a complex operation. The 90S precursor of the small 40S subunit undergoes a "molting" process, during which it progressively discards its outermost components.

Protein synthesis, programmed by the genetic information encoded in the DNA, is perhaps the most crucial process that takes place in biological cells. Proteins are indispensable for all organisms, because they are responsible for performing a vast range of biological functions. Indeed, the molecular machines that put proteins together—which are known as ribosomes—are themselves partly made up of specific proteins. The second vital ingredient of every ribosome is a small set of specific RNAs, which serve as scaffolds to which ribosomal proteins can be specifically attached. The synthesis of ribosomes is therefore an extremely complex, multistep process, which includes both assembly and maturation stages. This complexity explains why many of the details of the whole operation are still incompletely understood. Now a group of researchers led by Professor Roland Beckmann at LMU's Gene Center has obtained new insights into the maturation phase that gives rise to the small subunit of the functional ribosome in brewer's yeast. The study, which was carried out in collaboration with colleagues based in Heidelberg, appears in the journal Science.

In the cells of higher organisms, mature ribosomes are composed of two distinct subunits, each of which contains a long ribosomal RNA (rRNA) molecule (called 18S in the small and 25S in the large subunit in yeast). The subunits interact with one another and with the messenger RNAs that program the synthesis of each protein.

My comment: Consider that the biogenesis and maturation process of the first Ribosome had to emerge prebiotically, since the Ribosome is life essential. This is like the assembly of a complex machine, where each part must fit and must have interface compatibility. The Ribosome is a factory where many machine-like movements and actions occur in incredibly fast manner.   


For a working biological system to be built, the five following conditions would all have to be met:
1: Availability. Among the parts available for recruitment to form the system, there would need to be ones capable of performing the highly specialized tasks of individual parts, even though all of these items serve some other function or no function.
2: Synchronization. The availability of these parts would have to be synchronized so that at some point, either individually or in combination, they are all available at the same time.
3: Localization. The selected parts must all be made available at the same ‘construction site,’ perhaps not simultaneously but certainly at the time, they are needed.
4: Coordination. The parts must be coordinated in just the right way: even if all of the parts of a system are available at the right time, it is clear that the majority of ways of assembling them will be non-functional or irrelevant.
5: Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if subsystems or parts are put together in the right order, they also need to interface correctly.
( Agents Under Fire: Materialism and the Rationality of Science, pgs. 104-105 (Rowman & Littlefield, 2004). HT: ENV.)

Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. In other words : Why would natural selection select an intermediate biosynthesis product or a scaffold protein, which has by its own no use for the organism, unless there is a distant goal, as in this case, to make a ribosome? And that product keeps going through all necessary steps, up to the point to be ready to be assembled in a larger system ?  Never do we see blind, unguided processes leading to complex functional systems with integrated parts contributing to the overall design goal.


In yeast, the smaller 40S subunit is derived from a much larger precursor complex called the 90S pre-ribosome. The 90S precursor particle contains a single (35S) RNA molecule. The RNAs ultimately associated with each mature subunit are produced by the removal of specific internal and end-fragments. However, one of the segments the RNA found in the 90S precursor plays an important role in ensuring that the mature 18S rRNA in the 40S subunit folds into its correct three-dimensional form.

How the processing of the 35S rRNA is achieved has so far been unclear. The general idea was that, as the 40S subunit matures, the processing steps that give rise to the 18S rRNA take place, and the mature 40S particle eventually 'emerges' from the 90S precursor. The new study adds new details, which reveal that the process is rather more complicated than that. For a start, a specific enzyme (Dhr1) is required to ensure that the initial cleavage of the 35S rRNA precursor occurs at the right position. Dhr1 first exposes the cleavage site, enabling it to interact with the enzyme Utp24, which cuts the correct fragment off one end of the 35S rRNA.

Shedding takes place stepwise

In addition, the "emergence" of the 40S subunit entails an ordered series of reactions in which the outer shell of the 90S particle is progressively dissociated from the 40S. "It doesn't just go plop," Beckmann remarks. The process is actually reminiscent of the molting of an insect—shedding of the integument takes place layer by layer. "It's rather like those Russian dolls. When you open one, you find a smaller one nestled inside," says Beckmann. And with the aid of cryo-electron microscopy, the specialists in Munich were able to discriminate between the different three-dimensional complexes characteristic of each step in the process. Earlier biochemical experiments performed by a team at the Center for Biochemistry at Heidelberg University (BZH), led by Professor Ed Hurt, had already cast doubt on the previous en bloc model by providing evidence for the idea that shedding of the outer layers of the 90S particle took place stepwise.

The elucidation of such mechanisms is not only of interest from the point of view of basic research. As Beckmann points out, more and more disorders have been shown to be related to a lack of intact ribosomes. When errors occur in the assembly and maturation of these delicate and intricate molecular machines, they may ultimately lead to a relative death of ribosomes, which then perturbs the delicate equilibrium between protein synthesis and degradation. Among the resulting syndromes are diverse forms of muscle atrophy, growth anomalies, anemias and certain cancers.

My comment: How can someone deny that this is a goal oriented process? 

90S pre-ribosome transformation into the primordial 40S subunit

The eukaryotic small ribosomal subunit (40S) is the smaller subunit of the eukaryotic 80S ribosomes, with the other major component being the large ribosomal subunit (60S). Production of small ribosomal subunits initially requires the formation of a 90S precursor followed by an enigmatic process of restructuring into the primordial pre-40S subunit. .

1. the remodeling RNA helicase Dhr1 engages the 90S pre-ribosome, followed by


3. separating the 5′-external transcribed spacer (ETS) from 18S ribosomal RNA. Next,
4. the 5′-ETS and 90S assembly factors become dislodged, but this occurs sequentially, not en bloc.
5. Eventually, the primordial pre-40S emerges, still retaining some 90S factors including Dhr1, now ready to
6. unwind the final small nucleolar U3–18S RNA hybrid.

The principles of assembly and remodeling of large ribonucleoproteins requires transcription, processing, and modification of ribosomal RNA (rRNA) and the integration of ~80 ribosomal proteins. It is a  highly complex process which comprises more than 50 assembly factors. Many different actions have to be performed during the assembly process. These include

adoping
binding
cleaving
coordinating
cutting
closing
dissociating
dismantling
forming
processing,
interacting
integrating
interacting
interplaying
moving
processing
preventing premature recruitment
priming
replacing
rearranging
recruiting
remodeling
removing
relocating
releasing
relocating
replacing
shedding
stabilizing
stepwise reducing
translocating.

The huge 90S pre-ribosome transforms into its next major biogenesis intermediate, the primordial pre-40S, by large structural rearrangements, including the successive shedding of assembly factor modules. Several findings support the notion that the observed transition states are of physiological relevance and are not merely the products of random disintegration. Defects in ribosome biogenesis can have
drastic consequences for human health as ribosomopathies. Therefore, gaining more mechanistic insights into this elaborate maturation process and its integration into cellular signaling pathways is desirable.

My comment: Consider that the description refers just to the maturation and biogenesis of the small subunit of the Ribosome. Could such cutting edge, highly ordered and complex synthesis process have occurred prebiotically, by random unguided chemical reactions ? Consider that there was neither a genome present to instruct the information to make all the parts, and how the assembly could take place. And we are also facing a chicken egg problem. The ribosome is required to make the translation of the genetic information to make the polymers that constitute the Ribosome. What came first ? 





The Ribosomal Peptidyl Transferase Center: Structure, Function, Evolution, Inhibition
https://geneticacomportamento.ufsc.br/files/2013/08/Polacek05-Ribozima-peptidil-transferase.pdf
Claim: In the RNA World, what was the selective advantage for the host to have a proto-ribosome that “learned” to produce peptides and proteins? It is highly unlikely that the first catalyzed polypeptides themselves had any significant enzymatic functions. Noller (2004) therefore proposed that the driving force for the selection of primitive protein synthesis was to enlarge the structural and, hence, functional repertoire of RNA. e the first translation system that produced “functional” peptides did not evolve to pave an exit path out of the RNA world, but rather “aimed at” improving the properties of RNA molecules and ribozymes in the pre-protein world.
Reply: If that were the case, why do we not see this ocurring still today? Why would RNA's "seek" or "aim" to have functions at all, and more, on top of that, enlarge this functional repertoire? Molecules do not have the urge to function, or to become larger, or to complexify. Molecules will simply lay around, and desintegrate, depending on the environmental conditions.

Claim: This concept of ligand-induced RNA conformational changes obviously survived the transition from the RNA World to the contemporary DNA-RNA-protein world and represents the basic principle of riboswitch elements found in certain prokaryal mRNAs 
Reply: This is begging the question. There is no evidence that a transition from RNA to DNA is feasible. The better explanation is that an intelligent designer created the system fully developed, and able to start translation right from the beginning. 

Claim: However, the need for synchronization of the production and assembly of protodomains apparently favored their association (possibly via RNA ligation) into longer RNA molecules
Reply: Molecules do not have the urge to synchronize things. Only intelligent designers with distant goals do so. 

Claim: Higher-order complexes could have been formed that might have added functional diversity and sophistication to such a hypothetical proto-ribosome
Reply: The guesswork and just-so assertions in this paper are astonishing. Could have, might have. What about: Its extremely unlikely that unguided means produce sophisticated higher order complexes, because thats not how random molecules behave ?


The ribosome’s has highly efficient, or optimal, design features. Ribosomes are comprised of both protein and RNA molecules, and their proteins make up a sizable fraction of the total protein content of many cells. Cells contain many ribosomes, and naturally in order for the cell to duplicate, the ribosomes must be duplicated. This means a lot of protein synthesis must take place, in order to create all the proteins in all the ribosomes.
One way to help alleviate this production problem would be to have yet more ribosomes in the cell. But that would, in turn, create an even greater protein synthesis burden, since even more proteins would be needed for those additional ribosomes. One way to solve this conundrum is to use RNAs in ribosomes rather than proteins, where possible. It is a fascinating problem, and the paper concludes that we can understand the solution not as the result of evolutionary contingencies, but as a solution to a functional need: Rather than being relics of an evolutionary past, the unusual features of ribosomes may reflect an additional layer of functional optimization that acts on the collective properties of their parts. 2

Ribosomes are optimized for autocatalytic production 1
Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes—such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55–80 small, similarly sized segments—speed up their autocatalytic production. 

The ribosome doubling time places a hard bound on the cell doubling time, because for every additional ribosome to share the translation burden there is also one more to make . Even for the smallest and fastest ribosomes, it takes at least 6min, and typically much longer, for one ribosome to make a new set of r-proteins and this estimate does not account for the substantial time that is invested in the synthesis of ternary complexes. This bound seems to explain the observed limits on bacterial growth, because ribosomes must also spend much of their time making other proteins, and shows that ribosomes are under very strong selective pressure to minimize the time they spend reproducing. Similar principles might also apply to some eukaryotes, because the ribosomes of eukaryotes are larger and slower. In fact, even organisms in which cell doubling times are not limited by ribosome doubling times would benefit from faster ribosome production, allowing ribosomes to spend more of their time producing the rest of the proteome. This efficiency constraint was recently shown to have broad physiological consequences for cells. 

Journey Inside The Cell Start at 1:28
https://www.youtube.com/watch?v=1fiJupfbSpg&feature=emb_title

Next this RNA transcript approaches and passes through a molecular machine called the nuclear pore complex an information recognition device that controls the flow of information in and out of the cell's nucleus now we see the genetic assembly instructions on the messenger RNA approaching and arriving at a two-part chemical factory called a ribosome the site of protein synthesis as the messenger RNA transcript passes through the ribosome the process of translation begins during translation a mechanical assembly line builds a specifically sequence chain of amino acids in accord with the instructions on the transcript these amino acids are transported from other parts of the cell by molecules called transfer RNAs which link specific sequences of bases to corresponding amino acids the sequential arrangement of the amino acids determines the type of protein constructed

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes 30 June 2011 4
The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. The formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. The loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled preribosomes. 

The formation of eukaryotic ribosomes requires the production and assembly of four rRNAs and 80 ribosomal proteins. In Saccharomyces cerevisiae, this process begins with the RNA polymerase I-dependent transcription of the polycistronic 35S rRNA precursor in the nucleolus. This pre-RNA undergoes a series of modifications and cleavage steps to yield three of the four rRNAs present in mature ribosomes. The initial cleavage steps take place at the A0, A1, A2 and A3 sites of the 35S rRNA. The first three cleavages generate the 20S pre-rRNA, a precursor that is exported to the cytosol and cleaved to render the 18S rRNA of the small 40S ribosomal subunit. The cleavage at the A3 site generates the 27SA3 pre-rRNA that, upon further maturation, will yield the 5.8S and 25S rRNAs that will form part of the large 60S ribosomal subunit. These two processing pathways are not mutually dependent, since cleavage at A3 can precede cuts at A0, A1 and A2 sites. Concomitant (naturally accompanying or associated) to the cleavage events, the rRNAs assemble with ribosomal proteins and the 5S rRNA, a 60S subunit component that is independently transcribed by RNA polymerase III. All those processing and assembly reactions require numerous non-ribosomal factors that associate with pre-rRNAs in complexes known as pre-ribosomal particles . One of them is the 90S pre-ribosomal particle, also known as the small subunit (SSU) processome. In addition to the primary pre-rRNA, the U3 snoRNP and early assembled ribosomal proteins, this particle harbors ’50 non-ribosomal factors that play structural, regulatory and cleavage roles. Recent work has shed light on the recruitment mechanism employed to assemble some of those factors onto the nascent pre-rRNA. Different subsets of those proteins are grouped together in structurally autonomous subunits that pre-exist before the synthesis of the 35S pre-RNA and the formation of the 90S particle. These building blocks include the subunits known as tUTP (U3 protein complex required for transcription), UTP-B, UTP-C, Mpp10/Imp3/Imp4 and Bms1/Rcl1. tUTP (also known as UTP-A) is composed of seven proteins, and UTP-B and UTP-C both contain six proteins. Mmp10/Imp3/Imp4 and Bms1/Rcl1, as indicated by their names, are composed of three and two components, respectively. It has also been shown that some of those subunits, together with snoRNPs, associate with the pre-rRNA in a hierarchical and stepwise manner (Figure 1A). One of the first steps in the formation of the 90S pre-ribosome is the binding of the tUTP subunit to the nascent pre-rRNA, an event that is a condition sine qua non for the subsequent binding of other pre-ribosomal subunits and proteins. A large number of 90S particle components bind to this nucleation core following two mutually independent assembly branches. One branch involves the recruitment of Rrp5 and the subsequent incorporation of the UTP-C subunit in an Rrp5-dependent manner. In the second branch, the UTP-B subunit and the U3 snoRNP co-assemble onto the nucleation core to form a highly stable 90S particle intermediary that also contains the Mpp10/Imp3/Imp4 subcomplex, the GTPase Bms1 and other pre-ribosomal proteins. However, it is not known as yet whether those latter components associate to the pre-rRNA through assembly steps that are concurrent or downstream to the loading of the U3 snoRNP and the UTP-B subunits onto the 35S pre-rRNA. To get further insights into the assembly mechanism of the 90S pre-ribosomal particle, we decided to investigate in the present work the hierarchy of assembly of three 90S pre-ribosomal particle components, Imp4, Bms1 and Utp20. Imp4 is a RNA binding factor that forms a stable heterotrimeric subcomplex with Imp3 and Mpp10 (20,21). This subunit binds directly to the U3 snoRNA and is able to induce, in vitro, structural rearrangements in the U3 snoRNA that alter its base pairing with the pre-rRNA. Bms1 is another stable component of 90S pre-ribosomes that forms a small subcomplex with Rcl1. Bms1 is a GTPase that has been proposed to deliver Rcl1 to pre-ribosomes when bound to GTP. However, the timing and purpose of this regulated delivery is still poorly characterized. Finally, Utp20 is a HEAT-repeat protein present in both 90S and pre-40S particles. Although its function is unknown, we have previously hypothesized that it could be important for the building of early 90S particle intermediates because, according to proteomic and bioinformatics analyses, Utp20 seems to be heavily interconnected and in close physical proximity to components of the tUTP, UTP-B and UTP-C subunits.


Assembly hierarchy model of the 90S pre-ribosome. 
The binding of the tUTP subunit initiates the formation of the 90S particle and is required for the subsequent loading of the rest of the components. Two separate, and mutually independent, primary assembly steps have been identified. One of the steps involves the assembly of the U3 snoRNP and UTP-B subunits, and is required for the recruitment of at least 20 components of the particle, including the GTPase Bms1. The other primary assembly step is the binding of Rrp5, which is required for the subsequent incorporation of the UTP-C subunit. Bms1 is required for a secondary assembly step that drives the assembly of several proteins, including Utp20, Enp2, Kre33 and the Mpp10 subcomplex. This hierarchy of interactions has been established from this work and previous studies that analyzed the interdependence between factors for their binding to the 35S pre-rRNA, and the composition of pre-ribosomal complexes formed in the absence of specific proteins. In addition, available evidence indicates that Mrd1 and Sof1 might be primary assembly factors, and that Rok1 and Rrp36 are recruited through the Rrp5- and the UTP-B/ U3-dependent branches, respectively. Rcl1, a protein that forms a subcomplex with Bms1, might be recruited to early pre-ribosomes together with Bms1. However, there is still not enough evidence to place the Rcl1–Bms1 subunit in this hierarchical model. Dashed lines indicate the possibility of intermediate assembly steps. 


1. https://www.nature.com/articles/nature22998
2. https://evolutionnews.org/2017/08/national-association-of-biology-teachers-versus-the-ribosome/
3. https://sci-hub.ren/10.1126/science.abb4119
4. https://pdfs.semanticscholar.org/3ae2/48e0da3682b8191f3a2c29bcbb683a6c3610.pdf?_ga=2.117568398.643374322.1612298161-2000881064.1597437965

Quantum mechanic glimpse into peptide bond formation within the ribosome shed light on origin of life

https://reasonandscience.catsboard.com/t1661-translation-through-ribosomes-amazing-nano-machines#8027

https://link.springer.com/article/10.1007/s11224-017-0980-5

On origin of life
The high conservation of the proto-ribosome nucleotide sequence is suggestive of its robustness under diverse environmental conditions and hence hints at its prebiotic origin.

My comment: It is also evidence that a gradual, evolutionary, step-wise evolution of the sequence would be non-functional, and indication, that the Ribosome had to emerge from the start, fully functional and operational, "as is".

The Peptidyl Transferase Center  is located within an RNA apparatus, which possesses all of the capabilities required for peptide bond formation, it may be a vestige of the prebiotic world. Hence, we proposed that this conserved pocket-like region is a vestige of a prebiotic bonding entity, around which life has evolved. Hence, it seems that this is the primordial ribosome, and called the B proto-ribosome^. This seemingly remnant of the prebiotic era is still functioning in the heart of all of the contemporary ribosomes. The structure of this pocket, which seems to be ingeniously built for accommodating the 3′ ends of the A- and P-site tRNAs

The preservation of RNA activity in performing the extremely important process of genetic code translation indicates that RNA is capable of handling the complexity of the current cellular life, which requires a highly controlled sophisticated regulatory mechanism. Obviously, translation is much more complicated than accidental peptide bond formation. 

Remarkably, within the contemporary ribosomes, the distances between the regions involved in ribosome’s function are far beyond the possibility of any direct Bchemical talk^ (70–140 A). The symmetrical region is located at the heart of the ribosome and chemically connects to all of the ribosome functional centers involved in translation. Hence, it can transmit signals between them. 

This machinery is consistent with the idea that positioning of the reactive groups is the critical factor for catalysis, but does not exclude assistance from ribosomal or substrate moieties. Hence, by offering the frame for correct substrate positioning, as well as for catalytic contribution of the P-site tRNA 2′-hydroxyl group, it became evident that the ribosomal architectural frame governs the positional requirements and provides the means for substrate-mediated chemical catalysis.

There is a notion of the existence of an apparatus that could serve as the proto-ribosome, hypothesized to be a Bpocket-like^ RNA pseudo dimer that is capable of peptide bond formation, peptidyl transfer, and its elongation. Furthermore, the detection of similar twofold symmetryrelated regions in all known structures of the large ribosomal subunit not only indicates the universality of this mechanism but also emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation.

The transition state for formation of the peptide bond in the ribosome
https://www.pnas.org/content/103/36/13327

Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation.

The peptidyl transferase center (PTC), which is located at the depth of a cavity built primarily of ribosomal RNA. This cavity provides the remote interactions dominating initial substrate positioning with stereochemistry suitable for the motions associated with peptide-bond formation and nascent-chain elongation.  Despite the ribosome’s overall asymmetric structure, a sizable 2-fold symmetry-related region, relating RNA backbone fold and nucleotide orientations, rather than nucleotide sequence identities, was revealed around the PTC in all known ribosomes structures. The symmetry-related region contains ≈180 nt and extends far beyond the PTC. It connects all functional centers involved in amino acid polymerization, including peptide-bond formation, the focus of this article. This ribosomal elaborate architectural design guides the process of peptide-bond reaction by forcing a rotational motion consistent with the 2-fold rotation axis. The bond connecting the universally conserved single-strand tRNA–3′ end with the tRNA-acceptor stem of the A-site tRNA almost coincides with the symmetry axis, indicating that A- to P-site translocation involves two synchronized motions: an overall mRNA/tRNA sideways shift and a rotation of the tRNA 3′end. Guided by PTC components, the rotatory motion facilitates peptide-bond formation and nascent-chain elongation. Furthermore, this motion places the A-site nucleophilic amine and the P-site carbonyl carbon at a distance allowing for interactions with the P-site tRNA A76 O2′ throughout a significant part of the rotatory motion, consistent with its suggested participation in peptide-bond formation catalysis. The nascent proteins are directed by the rotatory motion into the exit tunnel at extended conformation, fitting the tunnel’s narrow opening. Hence, the ribosomal architecture provides all of the positional elements required for amino acid polymerization.

Paramount are the energetics of the formation of the transition state (TS), which governs the formation of the peptide bond. Here we show that we have been able to define a quantum mechanical transition state TS that is relevant to peptide-bond formation within the ribosome, characterize both its geometry and energy, and implicate these properties to events associated with peptide-bond formation and polypeptide elongations.

The key geometrical parameters of TS formation are summarized in Table 1, and Fig. 1 shows the image of the optimized TS geometry for the formation of the peptide bond in the ribosome.


Optimized peptide bond TS in the ribosome.
An initial geometry was obtained using the coordinates of ASM in D50S at the A site and its derived P-site tRNA. The amino acid of ASM was converted to alanine.

The optimized TS bond distances are labeled according to whether they are in the act of breaking or forming, to achieve the transition from reactants to products. The end result is that the peptide bond NC is formed, which leads to elongating the nascent protein. The new OH bond, which is formed on the P-site tRNA, saturates the open valence of the oxygen atom that would occur as the CO bond breaks to allow release of the amino acid transferred to the nascent protein. The remaining bond that is breaking in the TS, namely, NH, completes the release of the P-site tRNA. Hence, simultaneously with bond making and breaking, the former A-site tRNA can occupy the P site, which becomes available by the former P-site tRNA release.

Notably, the TS fits perfectly into the space available for the rotating A-site 3′ end, provided by the ribosome nucleotide surroundings within the ribosomal PTC (called here the “rotatory space”), after a 45° rotation of the A-site tRNA 3′ end toward the P site


The TS position within the volume occupied by the rotatory motion in the PTC of the ribosome.
(a) A schematic presentation of the combined linear and rotational motions involved in the passage of the A-site tRNA from the A site to the P site. The 2-fold symmetry axis is shown in red. The apparent overlap of the two tRNA stems is a result of the specific view (diagonal toward the back of the paper plane), chosen to show best the concerted motions.
(b) The approximately orthogonal view of the rotatory motion shown in a, looking approximately down the 2-fold symmetry axis, together with the TS, formed after 45° rotation (A site to P site) within the PTC of the ribosome. The transparent cyan “cloud” shows the entire rotatory space, as was simulated every 15° of the rotation. The ribosomal nucleotides are shown in gray. Those below the rotatory space are shown in lighter gray. The TS is shown in dark red. Nucleotides A2602 and U2585 are colored purple and yellow, respectively. Note the marked fit between the TS position and the space provided by the ribosome.
(c) Two views perpendicular to the 2-fold rotation axis. Shown are ends of tRNAs molecules at the P site (green), the A site (blue), the 2-fold axis (red), the TS (dark red), and the nucleotides C2452 and U2585 (gray). The TS lies at its best position between the A- and P-site tRNAs. (Left) View from the subunit interface. (Right) View from the PTC rear wall.

In all respects, it makes good chemical sense, in terms of formation of a peptide bond, the translocation of A-site tRNA to the P site, and P-site tRNA separation from the elongated chain. The chemical sense, after the mathematical criteria, is what corroborates the TS.  The TS is characterized mathematically by normal mode frequencies that are all positive, except for exactly one, which is negative, and corresponds to a vibration along the reaction coordinate sending the old reactants into the new products.

Quantum-Mechanical Study on the Mechanism of Peptide Bond Formation in the Ribosome
https://sci-hub.ren/https://pubs.acs.org/doi/full/10.1021/ja209558d

Interactions between active site residues and the 2′-OH are pivotal in orienting substrates in the active site for optimal catalysis. A second 2′-OH group  was identified to be crucial for peptide bond formation, namely that of A2451. The 2′- OH of A2451 was shown to be of potential functional importance.

The ribosome promotes the reaction of the amino acid condensation by properly orienting the reaction substrates.

Energy-dependent protein folding: modeling how a protein folding machine may work
https://www.biorxiv.org/content/10.1101/2020.09.01.277582v2.full.pdf

Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such protein folding machine might include direct manipulation of the peptide backbone. 

Considering the rotating motion of the tRNA 3’-end in the peptidyltransferase center of the ribosome, it is possible that this motion might introduce rotation to the nascent peptide and influence the peptide’s folding pathway in a way similar to what was observed in our simulations.

We performed molecular dynamics simulations in which a standard force field was augmented by the application of external mechanical forces to the polypeptide backbone. We compared these simulations to control runs without any additional external forces. The directional rotation of the Cterminal amino acid with simultaneous restriction of the movements of the N-terminal amino acid facilitated the formation of native structures in five diverse alpha-helical peptides, confirming that such constraints can have significant consequences for folding dynamics. Strikingly, application of mechanical force accelerated the folding of P4, a fragment of an on-pathway folding intermediate of the well-studied villin headpiece domain HP35, which is one of the fastest-folding protein domains known. The several-fold increase in the rate of P4 folding that was achieved in our experiments seems to suggest that the postulated “physical limit of folding” of HP35 as a whole could be overcome by a protein folding machine. The other four peptides in our experiments likewise attained their alpha-helical structure in the presence of the rotating force, but did not reach their native conformations when allowed to fold unassisted, even though we ran the control unassisted simulations for ~10 times longer than the simulations that included the application of the external force. Some of those peptides might take a very long time to reach their native conformations without application of an external force, whereas others might never fold unassisted, if their unfolded states are more stable than the folded conformations.

The 3’ terminus of the tRNA in the Asite of the ribosome peptidyl transferase center turns by nearly 180 degrees in every translation elongation cycle. Only a 45-degree swing is necessary to achieve the proper stereochemistry of the peptide bond formation; the function of the remaining portion of the turn is unknown, and we have hypothesized that it may be needed to facilitate co-translational folding

These results are in line with our protein folding machine hypothesis. They also support a hypothetical mechanism through which the machine would directly alter the conformations of proteins by applying mechanical force to the peptide backbone.  In vivo the peptide backbone can be manipulated into conformations that cannot be reached without assistance because they are either thermodynamically unstable or kinetically inaccessible. The results of our simulations thus demonstrate the feasibility of a protein folding machine. Some recently published results, including studies of the role of the exit tunnel in nascent chain folding

My comment: This demonstrates that protein folding ( which is essential to get functional proteins ) is a complex, finely orchestrated process that depends not only on the correct amino acid sequence, and the decrease in Gibbs free energy, but also an active energy-dependent process. The mechanism of action of the Ribosome protein folding machine  includes direct manipulation of the peptide backbone. To consider is the rotating motion of the tRNA 3’-end in the peptidyltransferase center of the ribosome. This motion introduces rotation to the nascent peptide which turns by nearly 180 degrees in every translation elongation cycle, and not only a 45-degree swing sufficient to achieve the proper stereochemistry of the peptide bond formation. That additional rotation seems to influence the peptide’s folding pathway in a way similar to what was observed in the simulations performed by the authors of above paper. 

The ability of any present-day protein to fold in isolation and without assistance not shared by most  proteins. Thus, the notion of an active, energy-dependent protein folding mechanism  in vivo reinforces the understanding of an intelligently bioengineered process  than the generally accepted evolutionary process, and that the ability of proteins to attain their native conformations must have evolved by natural selection of sequences that fold quickly and correctly (“evolution solved the protein folding problem” ) becomes more and more remotely possible. 

Cotranslational Protein Folding inside the Ribosome Exit Tunnel
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571824/

For small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. That the small zinc-finger domain ADR1a folds cotranslationally as the tether connecting it to the ribosome grows in length from ∼20 to ∼30 residues.  ADR1a buried deep in the vestibule of the exit tunnel, provides a clear demonstration that small proteins or protein domains can fold within the ribosome, as predicted by computational studies Although the zinc finger is one of the smallest independently folding protein domains, it has been estimated that ∼9% of all structural domains found in the PDB are less than 40 residues long, and ∼18% are less than 60 residues long. Folding of protein domains wholly or partly inside the exit tunnel may thus be not too uncommon, despite its relatively constrained geometry. 

Mutational analysis of protein folding inside the ribosome exit tunnel
https://febs.onlinelibrary.wiley.com/doi/full/10.1002/1873-3468.12504
The Ala scanning results are entirely consistent with in vitro studies of purified ADR1a: the residues most critical for stabilizing the folded state are the four Zn2+‐coordinating residues, and residues F12 and L18 that constitute the hydrophobic core.









Visualization by Cryo-EM of the ADR1a Domain in a Stalled Ribosome-ADR1a-SecM (Ms-Sup1; L = 25) Complex
(A) Schematic of the construct used for in vitro translation (top) and cryo-EM reconstructions of stalled E. coli ribosome-SecM-ADR1a complexes (left). The 30S subunit is depicted in yellow, the 50S subunit in gray, and the peptidyl-tRNA with the nascent polypeptide chain in green. Additionally, a cross-section through the cryo-EM density is shown in which the density for the nascent chain and the ADR1a domain (PDB: 2ADR) are depicted in green and red, respectively. A close-up of the tunnel and a schematic view are shown with the structure of the ADR1a domain fitted as rigid body depicted in red.
(B) Isolated density for the ADR1a domain (red) shown at different contour levels (top) compared with corresponding densities calculated from the NMR-derived molecular model of ADR1a (middle). Isolated cryo-EM density is shown transparent with the docked model (red) and the coordinated Zn2+ ion in yellow (bottom).

Protein refolding by the chaperones of the HSP70 family, may be also interpreted as evidence of protein folding in vivo being an active process

Realistic narratives of protein folding must therefore take into account the presence of a several exquisitely and masterfully planned molecular mechanisms that induce external forces and promote correct protein folding.

All living things rely on ribosomes, indicating that they must have been present when life began. Looking closely at the active site of the ribosome, where new proteins are built, the structure reveals that the machinery is composed of RNA, and a particular RNA base performs the reaction


Active site of the ribosome. 
This structure includes a ribosome with the tips of two transfer RNA molecules ( magenta and blue spheres ) bound in the protein-building site. The ribosome nucleotide shown in red catalyzes the reaction

Imprints of the genetic code in the ribosome
https://www.pnas.org/content/107/18/8298.full

The establishment of the genetic code remains elusive nearly five decades after the code was elucidated. The stereo chemical hypothesis postulates that the code developed from interactions between nucleotides and amino acids, yet supporting evidence in a biological context is lacking. We show here that anticodons are selectively enriched near their respective amino acids in the ribosome, and that such enrichment is significantly correlated with the canonical code over random codes.

Although multiple hypotheses have been proposed to explain why codons are selectively assigned to specific amino acids, empirical data are extremely rare and difficult to obtain leaving many theories in the realm of conjecture. Our results suggest that the essence of the primitive RNA-amino acid interaction remains at the heart of modern ribosomes

The Ribosome: Perfectionist Protein-Maker Trashes Error

http://reasonandscience.heavenforum.org/t1661-ribosomes-amazing-nano-machines


http://iaincarstairs.wordpress.com/2013/03/25/as-smart-as-molecules/

One machine common to all life on Earth is the ribosome.  Its strongly conserved nature, and the common sense observation that it makes everything else, indicates its central position in evolution.  The ribosome is not a single tool but a workshop split into two major parts, all created (using E. coli as an example) from around 7,400 amino acids, and around 250,000 atoms, all primed to use the strongest possible codon-amino acid mapping out of a practically endless range of possibilities.

Ribosomes can be so numerous as to make up 25% of the cell mass of E. coli. A striking feature of the ribosome is that, even given the large assorted collection of subunits, it self-assembles in vitro!

The core of the ribosome is RNA, supporting the idea that early forms of life relied on RNA rather than DNA.  But if such a workshop is necessary to create proteins, whether from templates of RNA or DNA – from where could the ribosome come from?  More vexing still for Darwinism is how editorial precision could arise in a system in which errors themselves were the key to prolific reproductive success at the start.  Why change a winning hand?

New discoveries are being made about the ribosome all the time.  Relevant to Darwinism, in 2009 Nature published some new discoveries by Johns Hopkins researchers concerning the remarkable actions of the ribosome’s ruthless quality control editor; if you think I tend to anthropomorphise molecules, note how the researchers detail -

   ..a new “proofreading step” during which the suite of translational tools called the ribosome recognizes errors, just after making them, and definitively responds by hitting its version of a “delete” button.

   It turns out.. ..that the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products which, as workhorses of the cell, carry out the very business of life.

   “What we now know is that in the event of miscoding, the ribosome cuts the bond and aborts the protein-in-progress, end of story,” says Rachel Green, a Howard Hughes Medical Institute investigator and professor of molecular biology and genetics in the Johns Hopkins University School of Medicine. “There’s no second chance.”

   “We thought that once the mistake was made, it would have just gone on to make the next bond and the next,” Green says. “But instead, we noticed that one mistake on the ribosomal assembly line begets another, and it’s this compounding of errors that leads to the partially finished protein being tossed into the cellular trash.”

   To their further surprise, the ribosome lets go of error-laden proteins 10,000 times faster than it would normally release error-free proteins, a rate of destruction Green says is “shocking” and reveals just how much of a stickler the ribosome is about high-fidelity protein synthesis.

   http://phys.org/news150559493.html#jCp




The translation process in the ribosome to occur, the ribosome must be able to proceed and go through the full translation sequence, it must be fully functional, no intermediate evolutionary stage will do it : beside this, it consists of two main subunits, ( beside a significant number of co-factors , which help in the build up process of the ribosome ) which makes it a irreducible complex system.

Replication most probably would not occur at pre-stage of a common ancestor, so evolution cannot be proposed as a driving factor at this stage.

lifeorigin::
RNA replication in the lab makes use of extensive investigator interference. Chemicals like amino acids, aldehydes, and sugars (other than ribose) are arbitrarily excluded. Very specific activation agents are used to encourage replication (ImpA for adenine, ImpG for guanine, ImpC for cytosine, and ImpU for uracil). The concentration of the chemicals (especially cytosine and ribose) is billions and billions of orders of magnitude higher than what one would expect under plausible prebiotic conditions.

Shajani Z :
Ribosome assembly needs the contributions of several assembly cofactors , including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit. These subunits would have no function of their own, why then would random processes produce them without a final goal and no forsight of function ?
Five following conditions would all have to be met in the biosynthesis process of the Ribosome:
Kairosfocus
C1: Availability. Among the parts available for recruitment to form a biological system consisting of multiple parts, there would need to be ones capable of performing the highly specialized tasks of the specific system, even though all of the items serve some other function or no function in another system where they were recruited from.
C2: Synchronization. The availability of these parts would have to be synchronized so that at some point, either individually or in combination, they are all available at the same time.
C3: Localization. The selected parts must all be made available at the same ‘construction site,’ perhaps not simultaneously but certainly at the time they are needed.
C4: Coordination.The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if the subunits  are put together in the right order, they also need to interface correctly.

The parts must be coordinated in just the right way: even if all of the parts of a ribosome are available at the right time, it is clear that the majority of ways of assembling them will be non-functional or irrelevant.
C5: Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if the subunits are put together in the right order, they also need to interface correctly.

Resumed : For the assembly of a biological system of multiple parts, following steps must be explained : the origin of the genome information to produce all subunits and assembly cofactors. Parts availability, synchronization, manufacturing and assembly coordination through genetic information, and interface compatibility. The individual parts must precisely fit together. All these steps are better explained through a super intelligent and powerful designer, rather than mindless natural processes by chance, or /  and evolution,  since we observe all the time minds capabilities producing  machines and factories, producing machines and end products.



http://www.hopkinsmedicine.org/news/media/releases/Lost_In_Translation

The enzyme machine that translates a cell's DNA code into the proteins of life is nothing if not an editorial perfectionist

Johns Hopkins researchers, reporting in the journal Nature January 7, have discovered a new "proofreading step" during which the suite of translational tools called the ribosome recognizes errors, just after making them, and definitively responds by hitting its version of a "delete" button.

It turns out, the Johns Hopkins researchers say, that the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products which, as workhorses of the cell, carry out the very business of life.

and it's this compounding of errors that leads to the partially finished protein being tossed into the cellular trash," she adds.

To their further surprise, the ribosome lets go of error-laden proteins 10,000 times faster than it would normally release error-free proteins, a rate of destruction that Green says is "shocking" and reveals just how much of a stickler the ribosome is about high-fidelity protein synthesis. "The cell is a wasteful system in that it makes something and then says, forget it, throw it out,"

That looks all ingeniously designed.......

http://www.nytimes.com/2009/10/08/science/08nobel.html?_r=0
Besides the implications for biomedical research, another consequence of the ribosome work was to resolve an old “classic chicken and egg problem” , Dr. Berg of the National Institute of General Medical Sciences explained. If ribosomes are needed to make proteins but they are also made of proteins, which came first?

J.Sarfati :
the DNA information requires a complex decoding machine, the ribosome, but the instructions to build ribosomes are on the DNA. And decoding requires energy from ATP, built by ATP-synthase motors, built from instructions in the DNA decoded by ribosomes … “vicious circles” for any materialistic origin theory, as leading philosopher of science Karl Popper put it .

http://newswire.rockefeller.edu/2013/08/14/structural-biologist-interested-in-ribosome-assembly-to-join-rockefeller-faculty/
What’s more, it’s something of a chicken-and-egg problem. “You need the machinery to be in place in order to manufacture proteins, but the machinery itself is made of proteins that must be manufactured,” Klinge says.

well, as far as i know without ribosomes there is no protein synthesis, without protein synthesis there is no life, without life there is no evolution so ribosomes cant come to existence via evolution so how did the form?

Facing these facts, i believe theists are justified to hold the position, that design explains best the origin of Ribosomes, and the origin of life.

[/quote]

well, as far as i know without ribosomes there is no protein synthesis, without protein synthesis there is no life, without life there is no evolution so ribosomes cant come to existence via evolution so how did the form?

The translation process in the ribosome to occur, the ribosome must be able to proceed and go through the full translation sequence, it must be fully functional, no intermediate evolutionary stage will do it : beside this, it consists of two main subunits, ( beside a significant number of co-factors , which help in the build up process of the ribosome ) which makes it a irreducible complex system.

Replication most probably would not occur at pre-stage of a common ancestor, so evolution cannot be proposed as a driving factor at this stage.

lifeorigin::
RNA replication in the lab makes use of extensive investigator interference. Chemicals like amino acids, aldehydes, and sugars (other than ribose) are arbitrarily excluded. Very specific activation agents are used to encourage replication (ImpA for adenine, ImpG for guanine, ImpC for cytosine, and ImpU for uracil). The concentration of the chemicals (especially cytosine and ribose) is billions and billions of orders of magnitude higher than what one would expect under plausible prebiotic conditions.

Shajani Z :Ribosome assembly needs the contributions of several assembly cofactors , including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit. These subunits would have no function of their own, why then would random processes produce them without a final goal and no forsight of function ?

Five following conditions would all have to be met:
Kairosfocus
C1: Availability. Among the parts available for recruitment to form the flagellum, there would need to be ones capable of performing the highly specialized tasks of paddle, rotor, and motor, even though all of these items serve some other function or no function.
C2: Synchronization. The availability of these parts would have to be synchronized so that at some point, either individually or in combination, they are all available at the same time.
C3: Localization. The selected parts must all be made available at the same ‘construction site,’ perhaps not simultaneously but certainly at the time they are needed.
C4: Coordination.
Besides the implications for biomedical research, another consequence of the ribosome work was to resolve an old “classic chicken and egg problem” about evolution,
J.Sarfati :
the DNA information requires a complex decoding machine, the ribosome, but the instructions to build ribosomes are on the DNA. And decoding requires energy from ATP, built by ATP-synthase motors, built from instructions in the DNA decoded by ribosomes … “vicious circles” for any materialistic origin theory, as leading philosopher of science Karl Popper put it .

http://newswire.rockefeller.edu/2013/08/14/structural-biologist-interested-in-ribosome-assembly-to-join-rockefeller-faculty/
What’s more, it’s something of a chicken-and-egg problem. “You need the machinery to be in place in order to manufacture proteins, but the machinery itself is made of proteins that must be manufactured,” Klinge says.

Shajani Z : :A ribosome consists of 50–70 different components and is, therefore, one of the most complicated structures known in biology. The large number of components requires a highly coordinated synthesis and assembly.

The parts must be coordinated in just the right way: even if all of the parts of a ribosome are available at the right time, it is clear that the majority of ways of assembling them will be non-functional or irrelevant.
C5: Interface compatibility. The parts must be mutually compatible, that is, ‘well-matched’ and capable of properly ‘interacting’: even if the subunits  are put together in the right order, they also need to interface correctly.

Resumed : For the assembly of a Ribosome, following steps must be explained : the origin of the genome information to produce all Ribosome subunits and assembly cofactors. Parts availability, synchronization, manufacturing and assembly coordination through genetic information, and interface compatibility. The individual parts must precisely fit together. All these steps are better explained through a super intelligent and powerful designer, rather than mindless natural processes by chance, or /  and evolution,  since we observe all the time minds capabilities producing ribosome-like machines and factories, producing machines and end products.

http://www.ncbi.nlm.nih.gov/pubmed/21529161
Shajani Z :
A ribosome consists of 50–70 different components and is, therefore, one of the most complicated structures known in biology. The large number of components requires a highly coordinated synthesis and assembly.


http://www.nobelprize.org/educational/medicine/dna/a/translation/ribosome_ass.html

This example of the Rate of Ribosome Synthesis is quite startling:

*HeLa cells (a type of human tumour cells) divide each 24 hours.
* Each cell contains around 10 million ribosomes, i.e. 7000 ribosomes are produced in the nucleolus each minute.
*Each ribosome contains around 80 proteins, i.e. more than 0.5 million ribosomal proteins are synthesised in the cytoplasm per minute.
*The nuclear membrane contains approximately 5000 pores. Thus, more than 100 ribosomal proteins are imported from the cytoplasm to the nucleus per pore and minute. At the same time 3 ribosomal subunits are exported from the nucleus to the cytoplasm per pore and minute.




http://sws1.bu.edu/mfk/ribosome.pdf




Althougheven this basic pathway is very complicated, translation involves many other features that have also been the subject of structural and functional studies in recent years. These include the rescue of stalled ribosomes, programmed frameshifting, the interaction of the nascent peptide with the exit tunnel, the modification of the peptide as it emerges from the ribosome, its folding and its transport across or insertion into membranes, and the regulation of translation. the extremely complicated field of eukaryotic translation, especially initiation, is sure to be increasingly targeted by biophysical and biochemical techniques.

Overview of bacterial translation. For simplicity, not all intermediate steps are shown. The colour scheme shown here is used consistently throughout this review. aa-tRNA, aminoacyl-tRNA; EF elongation factor; IF, initiation factor; RF, release factor.


Ribosomes composed of two subunits

3d:
http://www.rcsb.org/pdb/explore/jmol.do?structureId=2WDK&split=yes&asymIds=2WDK%2C2WDL%2C2WDM%2C2WDN&bionumber=1

A Ribosome is composed of two subunits, made of RNA chains with proteins bound on the outside.  The molecular movements of this complex provides a specific catalyst for the creation of amino-acid polymers.   These biological nanomachines are the 3D printers of the cell, producing thousands of different proteins.

http://cellmorphs.tumblr.com/

A Ribosome is composed of two subunits, made RNA chains and proteins bound on its outside.  The molecular movements of this complex provide a very specific catalyst for the creation of amino-acid polymers.   These biological nanomachines are the 3D printers of the cell, producing thousands of different proteins.

Ahh, the mighty ribosome. A biological machine comprised of an elaborate conglomeration of intricately-folded proteins and RNAs, none capable of building anything on its own, but together creating nature’s most advanced piece of chemical machinery.

It’s a fascinating chicken-and-egg problem written in nucleic and amino acids, a thing that has to exist in order to make itself

It’s also a thing that has to exist to make any of us, the translator of the genetic code, taking the instructions for life and assembling the things that do stuff inside of all of life.

When I look at this, I see the incredible beauty of evolution Gods creation  written in chemistry.

http://cshperspectives.cshlp.org/content/4/4/a003681.full
The molecular evolution of translation poses at least three difficult questions: (1) The chicken-or-the-egg problem: if the ribosome requires proteins to function, where did the proteins come from to make the first ribosome? (2) What was the driving force for evolution of the ribosome? and (3) How did coding arise? Thanks to numerous advances in this field, we now have a likely answer to the first question and a plausible basis for answering the second. Despite many decades of thinking about the third question, the origins of coding remain a puzzle. Another question, implicit in the RNA World hypothesis, is (4) Can we account for...

Leading Biologists Marvel at the "Irreducible Complexity" of the Ribosome, but Prefer Evolution-of-the-Gaps

Professor of Genetics at Harvard Medical School and Director of the Center for Computational Genetics, similarly marveled at the complexity of the ribosome:

The ribosome, both looking at the past and at the future, is a very significant structure,  it's the most complicated thing that is present in all organisms.It can change from DNA three nucleotides into one amino acid. That's really marvelous. We need to understand that better.

Craig Venter suggested that by sequencing the genomes of more organisms perhaps we could reconstruct a primitive precursor ribosome. But Church is skeptical that this is unlikely to help because current biology reveals that a minimum number of genes are required for a functional ribosome--and that minimum number is still quite large:

But isn't it the case that, if we take all the life forms we have so far, isn't the minimum for the ribosome about 53 proteins and 3 polynucleotides? And hasn't that kind of already reached a plateau where adding more genomes doesn't reduce that number of proteins?

The conversation that follows is striking, showing that as far as we know, the ribosome has "irreducibly complexity":

   VENTER: Below ribosomes, yes: you certainly can't get below that. But you have to have self-replication.

   CHURCH: But that's what we need to do -- otherwise they'll call it irreducible complexity. If you say you can't get below a ribosome, we're in trouble, right? We have to find a ribosome that can do its trick with less than 53 proteins.

   VENTER: In the RNA world, you didn't need ribosomes.

   CHURCH: But we need to construct that. Nobody has constructed a ribosome that works well without proteins.

   VENTER: Yes.

   SHAPIRO: I can only suggest that a ribosome forming spontaneously has about the same probability as an eye forming spontaneously.

   CHURCH: It won't form spontaneously; we'll do it bit by bit.

   SHAPIRO: Both are obviously products of long evolution of preexisting life through the process of trial and error.

   CHURCH: But none of us has recreated that any.

   SHAPIRO: There must have been much more primitive ways of putting together

   CHURCH: But prove it.

We don't know how the ribosome and its required proteins evolved, but we know that "Both are obviously products of long evolution of preexisting life through the process of trial and error." This is a prime example of "evolution-of-the-gaps,"

CNC Machining and One Ruthless Editor

The ribosome is not a single tool but a workshop split into two major parts, all created  from around 7,400 amino acids ( E. coli ), and around 250,000 atoms, all primed to use the strongest possible codon-amino acid mapping out of a practically endless range of possibilities.

Ribosomes can be so numerous as to make up 25% of the cell mass of E. coli. A striking feature of the ribosome is that, even given the large assorted collection of subunits, it self-assembles in vitro!

The core of the ribosome is RNA, supporting the idea that early forms of life relied on RNA rather than DNA.  But if such a workshop is necessary to create proteins, whether from templates of RNA or DNA – from where could the ribosome come from?  More vexing still for Darwinism is how editorial precision could arise in a system in which errors themselves were the key to prolific reproductive success at the start.  Why change a winning hand?

New discoveries are being made about the ribosome all the time.  In 2009 Nature published some new discoveries by Johns Hopkins researchers concerning the remarkable actions of the ribosome’s ruthless quality control editor; if you think I tend to anthropomorphise molecules, note how the researchers detail -

..a new “proofreading step” during which the suite of translational tools called the ribosome recognizes errors, just after making them, and definitively responds by hitting its version of a “delete” button.

It turns out.. ..that the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products which, as workhorses of the cell, carry out the very business of life.

“What we now know is that in the event of miscoding, the ribosome cuts the bond and aborts the protein-in-progress, end of story,” says Rachel Green, a Howard Hughes Medical Institute investigator and professor of molecular biology and genetics in the Johns Hopkins University School of Medicine. “There’s no second chance.”

“We thought that once the mistake was made, it would have just gone on to make the next bond and the next,” Green says. “But instead, we noticed that one mistake on the ribosomal assembly line begets another, and it’s this compounding of errors that leads to the partially finished protein being tossed into the cellular trash.”

To their further surprise, the ribosome lets go of error-laden proteins 10,000 times faster than it would normally release error-free proteins, a rate of destruction Green says is “shocking” and reveals just how much of a stickler the ribosome is about high-fidelity protein synthesis.

https://web.archive.org/web/20130503211721/http://iaincarstairs.wordpress.com/2013/03/25/as-smart-as-molecules/

Ribosome of escheria coli:

In eubacteria and archaea the large subunit comprises ~3,000 nucleotides of ribosomal RNA (rRNA), > 30 proteins and sediments at 50S, whereas the ~1,500 nucleotides and > 20 proteins of the smaller subunit sediment at 30S. These conveniently different sedimentation coefficients are used as descriptors of the large (50S) and the small (30S) ribosomal subunits, while the entire ribosomal particle is referred to as the 70S ribosome (in eukaryotes these are 60S, 40S and 80S, respectively).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783306/

Subunit composition of ribosome 
[(RrsA)(RpsA)(RpsB)(RpsC)(RpsD)(RpsE)(RpsF)(RpsG)(RpsH)(RpsI)(RpsJ)(RpsK)(RpsL)(RpsM)(RpsN)(RpsO)(RpsP)(RpsQ)(RpsR)(RpsS)(RpsT)(RpsU)(Sra)][(RrlA)(RrfA)(RplA)(RplB)(RplC)(RplD)(RplE)(RplF)([RplJ][(RplL)₂]₂)(RplI)(RplK)(RplM)(RplN)(RplO)(RplP)(RplQ)(RplR)(RplS)(RplT)(RplU)(RplV)(RplW)(RplX)(RplY)(RpmA)(RpmB)(RpmC)(RpmD)(RpmE)(RpmF)(RpmG)(RpmH)(RpmI)(RpmJ)]

30S ribosomal subunit 
(RrsA)(RpsA)(RpsB)(RpsC)(RpsD)(RpsE)(RpsF)(RpsG)(RpsH)(RpsI)(RpsJ)(RpsK)(RpsL)(RpsM)(RpsN)(RpsO)(RpsP)(RpsQ)(RpsR)(RpsS)(RpsT)(RpsU)(Sra) (summary available)
16S ribosomal RNA = RrsA (extended summary available)
30S ribosomal subunit protein S1 = RpsA (extended summary available)
30S ribosomal subunit protein S2 = RpsB (summary available)
30S ribosomal subunit protein S3 = RpsC (summary available)
30S ribosomal subunit protein S4 = RpsD (extended summary available)
30S ribosomal subunit protein S5 = RpsE (extended summary available)
30S ribosomal subunit protein S6 = RpsF (extended summary available)
30S ribosomal subunit protein S7 = RpsG (extended summary available)
30S ribosomal subunit protein S8 = RpsH (extended summary available)
30S ribosomal subunit protein S9 = RpsI (extended summary available)
30S ribosomal subunit protein S10 = RpsJ (extended summary available)
30S ribosomal subunit protein S11 = RpsK (extended summary available)
30S ribosomal subunit protein S12 = RpsL (extended summary available)
30S ribosomal subunit protein S13 = RpsM (extended summary available)
30S ribosomal subunit protein S14 = RpsN (summary available)
30S ribosomal subunit protein S15 = RpsO (extended summary available)
30S ribosomal subunit protein S16 = RpsP (extended summary available)
30S ribosomal subunit protein S17 = RpsQ (summary available)
30S ribosomal subunit protein S18 = RpsR (extended summary available)
30S ribosomal subunit protein S19 = RpsS (summary available)
30S ribosomal subunit protein S20 = RpsT (extended summary available)
30S ribosomal subunit protein S21 = RpsU (summary available)
30S ribosomal subunit protein S22 = Sra (summary available)

50S ribosomal subunit = 
(RrlA)(RrfA)(RplA)(RplB)(RplC)(RplD)(RplE)(RplF)([RplJ][(RplL)₂]₂)(RplI)(RplK)(RplM)(RplN)(RplO)(RplP)(RplQ)(RplR)(RplS)(RplT)(RplU)(RplV)(RplW)(RplX)(RplY)(RpmA)(RpmB)(RpmC)(RpmD)(RpmE)(RpmF)(RpmG)(RpmH)(RpmI)(RpmJ)
23S ribosomal RNA = RrlA (extended summary available)
5S ribosomal RNA = RrfA (extended summary available)
50S ribosomal subunit protein L1 = RplA (extended summary available)
50S ribosomal subunit protein L2 = RplB (summary available)
50S ribosomal subunit protein L3 = RplC (summary available)
50S ribosomal subunit protein L4 = RplD (extended summary available)
50S ribosomal subunit protein L5 = RplE (extended summary available)
50S ribosomal subunit protein L6 = RplF (extended summary available)
50S ribosomal protein complex L8 = (RplJ)([RplL]₂)₂ (summary available)
50S ribosomal subunit protein L10 = RplJ (extended summary available)
50S ribosomal subunit protein L7/L12 dimer = (RplL)₂
50S ribosomal subunit protein L12 = RplL
50S ribosomal subunit protein L9 = RplI (extended summary available)
50S ribosomal subunit protein L11 = RplK (extended summary available)
50S ribosomal subunit protein L13 = RplM (extended summary available)
50S ribosomal subunit protein L14 = RplN (extended summary available)
50S ribosomal subunit protein L15 = RplO (summary available)
50S ribosomal subunit protein L16 = RplP (extended summary available)
50S ribosomal subunit protein L17 = RplQ (summary available)
50S ribosomal subunit protein L18 = RplR (extended summary available)
50S ribosomal subunit protein L19 = RplS (extended summary available)
50S ribosomal subunit protein L20 = RplT (extended summary available)
50S ribosomal subunit protein L21 = RplU (summary available)
50S ribosomal subunit protein L22 = RplV (extended summary available)
50S ribosomal subunit protein L23 = RplW (extended summary available)
50S ribosomal subunit protein L24 = RplX (extended summary available)
50S ribosomal subunit protein L25 = RplY (extended summary available)
50S ribosomal subunit protein L27 = RpmA (extended summary available)
50S ribosomal subunit protein L28 = RpmB (summary available)
50S ribosomal subunit protein L29 = RpmC (summary available)
50S ribosomal subunit protein L30 = RpmD (summary available)
50S ribosomal subunit protein L31 = RpmE (extended summary available)
50S ribosomal subunit protein L32 = RpmF (summary available)
50S ribosomal subunit protein L33 = RpmG (extended summary available)
50S ribosomal subunit protein L34 = RpmH (extended summary available)
50S ribosomal subunit protein L35 = RpmI (summary available)
50S ribosomal subunit protein L36 = RpmJ (extended summary available)

Initial work in the bacterium Escherichia coli identified the ribosome-interacting protein elongation factor P to be essential for resolving translation stalling at proline stretches. In eukaryotes, the homologous factor eIF5A is a highly abundant35 and essential protein that is comprised of only 157 amino acids and contains a unique post-translational modification called hypusine [G].
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054806/

Escherichia coli K-12 substr. MG1655 All-Genes Class: ribosomes
https://biocyc.org/ECOLI/NEW-IMAGE?type=ECOCYC-CLASS&object=BC-6.6

dbpA (ATP-dependent RNA helicase DbpA),
deaD (ATP-dependent RNA helicase DeaD),
der (50S ribosomal subunit stability factor),
ffs (signal recognition particle 4.5S RNA),
hflX (ribosome rescue factor HflX),
raiA (ribosome-associated inhibitor A),
rbfA (30S ribosome binding factor),
relA (GDP/GTP pyrophosphokinase),
rimM (ribosome maturation factor RimM),
rimP (ribosome maturation factor RimP),
rmf (ribosome modulation factor),
rplA (50S ribosomal subunit protein L1),
rplB (50S ribosomal subunit protein L2),
rplC (50S ribosomal subunit protein L3),
rplD (50S ribosomal subunit protein L4),
rplE (50S ribosomal subunit protein L5),
rplF (50S ribosomal subunit protein L6),
rplI (50S ribosomal subunit protein L9),
rplJ (50S ribosomal subunit protein L10),
rplK (50S ribosomal subunit protein L11),
rplL (50S ribosomal subunit protein L7),
rplM (50S ribosomal subunit protein L13),
rplN (50S ribosomal subunit protein L14),
rplO (50S ribosomal subunit protein L15),
rplP (50S ribosomal subunit protein L16),
rplQ (50S ribosomal subunit protein L17),
rplR (50S ribosomal subunit protein L18),
rplS (50S ribosomal subunit protein L19),
rplT (50S ribosomal subunit protein L20),
rplU (50S ribosomal subunit protein L21),
rplV (50S ribosomal subunit protein L22),
rplW (50S ribosomal subunit protein L23),
rplX (50S ribosomal subunit protein L24),
rplY (50S ribosomal subunit protein L25),
rpmA (50S ribosomal subunit protein L27),
rpmB (50S ribosomal subunit protein L28),
rpmC (50S ribosomal subunit protein L29),
rpmD (50S ribosomal subunit protein L30),
rpmE (50S ribosomal subunit protein L31),
rpmF (50S ribosomal subunit protein L32),
rpmG (50S ribosomal subunit protein L33),
rpmH (50S ribosomal subunit protein L34),
rpmI (50S ribosomal subunit protein L35),
rpmJ (50S ribosomal subunit protein L36),
rpsA (30S ribosomal subunit protein S1),
rpsB (30S ribosomal subunit protein S2),
rpsC (30S ribosomal subunit protein S3),
rpsD (30S ribosomal subunit protein S4),
rpsE (30S ribosomal subunit protein S5),
rpsF (30S ribosomal subunit protein S6),
rpsG (30S ribosomal subunit protein S7),
rpsH (30S ribosomal subunit protein S8),
rpsI (30S ribosomal subunit protein S9),
rpsJ (30S ribosomal subunit protein S10),
rpsK (30S ribosomal subunit protein S11),
rpsL (30S ribosomal subunit protein S12),
rpsM (30S ribosomal subunit protein S13),
rpsN (30S ribosomal subunit protein S14),
rpsO (30S ribosomal subunit protein S15),
rpsP (30S ribosomal subunit protein S16),
rpsQ (30S ribosomal subunit protein S17),
rpsR (30S ribosomal subunit protein S18),
rpsS (30S ribosomal subunit protein S19),
rpsT (30S ribosomal subunit protein S20),
rpsU (30S ribosomal subunit protein S21),
rrfA (5S ribosomal RNA),
rrfB (5S ribosomal RNA),
rrfC (5S ribosomal RNA),
rrfD (5S ribosomal RNA),
rrfE (5S ribosomal RNA),
rrfF (5S ribosomal RNA),
rrfG (5S ribosomal RNA),
rrfH (5S ribosomal RNA),
rrlA (23S ribosomal RNA),
rrlB (23S ribosomal RNA),
rrlC (23S ribosomal RNA),
rrlD (23S ribosomal RNA),
rrlE (23S ribosomal RNA),
rrlG (23S ribosomal RNA),
rrlH (23S ribosomal RNA),
rrsA (16S ribosomal RNA),
rrsB (16S ribosomal RNA),
rrsC (16S ribosomal RNA),
rrsD (16S ribosomal RNA),
rrsE (16S ribosomal RNA),
rrsG (16S ribosomal RNA),
rrsH (16S ribosomal RNA),
rsgA (ribosome small subunit-dependent GTPase A),
sra (30S ribosomal subunit protein S22),
srmB (ATP-dependent RNA helicase SrmB),
ssrA (tmRNA),
yggL (putative ribosome assembly factor YggL),
yhbY (ribosome assembly factor YhbY)

16S ribosomal RNA 

It has several functions:
Like the large (23S) ribosomal RNA, it has a structural role, acting as a scaffold defining the positions of the ribosomal proteins.
The 3′-end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNA. The 3′-end of 16S RNA binds to the proteins S1 and S21 known to be involved in initiation of protein synthesis[5]
Interacts with 23S, aiding in the binding of the two ribosomal subunits (50S and 30S)
Stabilizes correct codon-anticodon pairing in the A-site, via a hydrogen bond formation between the N1 atom of adenine residues 1492 and 1493 and the 2′OH group of the mRNA backbone

First, 16S rRNA is present in all known prokaryotic organisms. Second, it is poorly subjected to lateral gene transfer. Third, the extremely conserved scaffolding by ribosomal proteins makes its sequence extremely conserved in certain regions, while other regions not directly involved in the stabilization and exposed to solvent are relatively free from evolutionary constraints, so they were extremely variable . In total, we can count nine variable regions (V1 to V9) of different size in the approximately 1500 bp constituting a full 16S rRNA molecule. Targeting all or some of these regions has been considered in the last two decades the gold standard for phylogenetic studies of microbial communities, as well as for assigning taxonomic names to prokaryotes, both archaea and bacteria.


Perspectives on prokaryote’s ribosome.
(A) Two opposite sides of the 3D structure of the prokaryotic ribosome with ribosomal proteins, 23S rRNA, 16S rRNA and 5S rRNA in white, green, red and blue, respectively.
(B) Two opposite sides of the 3D structure of the prokaryotic ribosome small subunit (SSU) with ribosomal proteins and 16S rRNA in white and red, respectively.
(C) Localization and color-coding of the nine 16S variable regions in the 3D structure of a scaffolded 16S rRNA devoid of ribosomal proteins. 
(D) Localization of the nine, color-coded as in C, 16S variable regions in the classical two-dimensional representation of the 16S rRNA secondary structure from E. coli.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288201/


16S rRNA secondary structure, showing conserved parts for Prokaryotes (IUPAC letters) and amongst Archea/Bacteria (asterisks) all others as dots. Adapted (in SVG!) from Woese Bacterial evolution 1987.

30S ribosomal subunit protein S1
S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. It has a pivotal role in stabilizing the mRNA on the ribosome. 3 In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding center.


Location of S1 within the 30S subunit.
(a) Surface representation of the 11.5-Å resolution cryo-EM map.
(b) X-ray structure of the 30S subunit, filtered to the resolution of the cryo-EM map and shown in the same solvent-side orientation. The area highlighted with a rectangle shows a large, extra mass of density in the cryo-EM map (a). (c and d) The difference map (red), obtained by subtracting the masses corresponding to only 30S ribosomal proteins in the x-ray and cryo-EM maps, is superimposed on the cryo-EM map (c) and on the filtered x-ray map of the 30S subunit
(d). In c, the 30S map is shown as a semitransparent surface, in which the difference density map is embedded. The S21 mass is marked with an arrow in c and d.

Protein S1 is the largest ribosomal protein, 68 kDa, present in the small subunit of the Escherichia coli 70S ribosome.  Protein S1 has been reported to be necessary in some cases for translation initiation and for translation elongation. It is the only ribosomal protein that has a high affinity for mRNA. As a ribosomal protein, S1 is strikingly atypical. 






1. https://en.wikipedia.org/wiki/16S_ribosomal_RNA
2. https://www.sciencedirect.com/science/article/pii/B9780081022689000057
3. Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA
https://www.pnas.org/content/98/21/11991
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288201/

Protein folding, surprising mechanisms point to an arranged set up

https://reasonandscience.catsboard.com/t1661p25-translation-through-ribosomes-amazing-nano-machines#8052

Prebiotic protein folding is indeed a huge problem, since we know now that the ribosome has a helping hand in promoting the right folding. And when that does not occur, no deal. So you need a ribosome to have functional protein folds, but you need functional folds to make a ribosome. What came first ?

Proteins, in order to become functional, must fold into very specific 3D shapes, which happens right when they come out of the ribosome, where they are synthesized. Specific protein shape and conformation depends on the interactions between its amino acid side chains. For a protein to function it must fold into a resting state which is a complex three-dimensional structure.  If a protein fails to fold into its functional structure then it is not only without function but it can become toxic to the cell. As proteins fold, they test a variety of conformations before reaching their final form, which is unique and compact. Folded proteins are stabilized by thousands of noncovalent bonds between amino acids. A relatively small protein of only 100 amino acids can take some 10^100 different configurations. If it tried these shapes at the rate of 100 billion a second, it would take longer than the age of the universe to find the correct one. Just how these molecules do the job in nanoseconds, nobody knows. 1

If we take one of the smallest free living bacteria, they have about 1300 proteins, with an average of about 400 amino acids. If their amino acid sequence and arrangement were to emerge prebiotically, without the instructional information from a genome, more attempts would be required  than the number of atoms in the universe, to get the right set. Not considering all the inherent problems with this scenario, like the lack of mechanisms to select the right amino acid set used in life, and sorting out of the right-handed ones ( life uses only left-handed amino-acids ), the fact that some amino acids never have been found besides being synthesized in the Cell, this is a major problem. And so the fact, that the linking bonds of these polymers are peptide and ester bonds. In both cases, the polymerization reaction is thermodynamically uphill, with hydrolysis being favored. ( hydrolysis  means that any chemical reaction in which a molecule of water ruptures one or more chemical bonds )  How then can polymers be synthesized? For prebiotic scenarios, there is no compelling answer. In the cell, the monomers have been chemically activated by an input of metabolic energy so that polymerization is spontaneous in the presence of ribosomes that catalyze polymerization.  Catalyzing bond formation in the Ribosome comes out to be a very carefully engineered and precise process, where the surrounding ribonucleotides must be placed very precisely, at the right place. How do proteins fold from a linear sequence of amino acids into functional form, where they can operate as molecular machines ? 

Science has unraveled surprising and astonishing details of what mechanisms might be in place, answering this question. 

One of the most important mechanisms that play a vital role in the cell is the amino acid peptide bond formation during protein synthesis. It takes place in the so-called peptidyl transferase center, which is the reaction center in the Ribosome.  Peptide bond formation is by no means a simple, or trivial task. The process is so intriguingly complex, that a science paper in 2015 had still to admit that:  The process of peptide bond formation is of particular importance, being the heart of protein synthesis. 1 The detailed mechanism of peptidyl transfer, as well as the atoms and functional groups involved in this process are still in limbo. 

The ribosome speeds up the reaction rate and catalyzes peptide bond formation 10 million times faster than  compared to an uncatalyzed reaction.  The ribosome subunit, where the catalysis takes place, is called 23S ribosomal RNA. It has a length of  2904 nucleotides(in E. coli). 

A precise, minutely orchestrated arrangement of just two main players amongst these 2904 nucleotides is absolutely essential,  the interaction of ribose 2'-OH at position A2451 , and the 2’ hydroxyl of the P site substrate A76  . They are pivotal in orienting substrates in the active site for optimal catalysis, and play a key role in polypeptide bond formation. 

My comment: Consider this as an extraordinary engineering feat. Amongst 2905 nucleotides, just one is the main player interacting with another to promote this life essential reaction, and had to be positioned precisely in the right spot. 

Evidently, the positioning of all substrates, transition states, and ribosomal residues contributing to the concerted redistribution of charges must be tightly controlled to achieve efficient transpeptidation compatible with the observed in vivo rates of amino acid polymerization of about 20 amino acids per second. 

Molecular biology of the Cell, Alberts, 6th ed. pg. 369

Producing an overall speed of translation of 20 amino acids incorporated per second in bacteria. Mutant bacteria with a specific alteration in the small ribosomal subunit have longer delays and translate mRNA into protein with an accuracy considerably higher than this; however, protein synthesis is so slow in these mutants that the bacteria are barely able to survive.

Question: How could the right speed have been obtained with trial and error, if slow mutants do not survive? Had the speed not to be right from the beginning?

This 2'-OH renders almost full catalytic power. These data highlight the unique functional role of the A2451 2'-OH for peptide bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site. Key in this reaction is the presence of a proton shuttling group.  The observed 100-fold reduction in the reaction rate by mutation of P-site A76 20-OH group  is indication of this group's activity during the peptidyl transfer reaction.

Remember this functional group, A2451. I will return to it at the end of this article.

Remarkably, as we will see in the following, protein folding is not only dependent on the amino acid sequence, or stabilizing forces. 

A paper reports:
Protein folding in living cells requires a mechanism of action through direct manipulation of the peptide backbone during polypeptide bond formation.  Considering the rotating motion of the tRNA 3’-end in the peptidyltransferase center of the ribosome, it is possible that this motion might introduce rotation to the nascent peptide and influence the peptide’s folding pathway. The 3’ terminus of the tRNA in the A-site of the ribosome peptidyl transferase center turns by nearly 180 degrees in every translation elongation cycle. Only a 45-degree swing is necessary to achieve the proper stereochemistry of the peptide bond formation; the function of the remaining portion of the turn is hypothesized to be needed to facilitate co-translational folding 1

Experimental results are in line with our  hypothetical mechanism through which the ribosome  directly altesr the conformations of proteins by applying mechanical force to the peptide backbone.  In vivo the peptide backbone can be manipulated into conformations that cannot be reached without assistance because they are either thermodynamically unstable or kinetically inaccessible. The results of our simulations thus demonstrate the feasibility of a protein folding mechanism during peptide bond formation.

My comment: This demonstrates that protein folding ( which is essential to get functional proteins ) is a complex, finely orchestrated process that depends not only on the correct amino acid sequence, and the decrease in Gibbs free energy, but also an active energy-dependent process. The mechanism of action of the ribosome (protein folding machine ). That means, without the concerted action of the ribosome, the original minimal proteome would never have formed prebiotically in absence of the ribosome, directly involved in the folding process.  The ability of any present-day protein to fold in isolation and without assistance not shared by most  proteins. Thus, the notion of an active, energy-dependent protein folding mechanism  in vivo reinforces the understanding of an intelligently bioengineered process  than the generally accepted evolutionary process, and that the ability of proteins to attain their native conformations must have evolved by natural selection of sequences that fold quickly and correctly (“evolution solved the protein folding problem” ) becomes more and more remotely possible. 

And more mechanisms are in play helping protein folding insider the ribosome:  
Some recently published results, include studies of the role of the exit tunnel in nascent chain folding.  For small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. That the small zinc-finger domain ADR1a folds cotranslationally as the tether connecting it to the ribosome grows in length from ∼20 to ∼30 residues.  ADR1a buried deep in the vestibule of the exit tunnel, provides a clear demonstration that small proteins or protein domains can fold within the ribosome, as predicted by computational studies Although the zinc finger is one of the smallest independently folding protein domains, it has been estimated that ∼9% of all structural domains found in the PDB are less than 40 residues long, and ∼18% are less than 60 residues long. Folding of protein domains wholly or partly inside the exit tunnel may thus be not too uncommon, despite its relatively constrained geometry. But even MORE REMARKABLY: In the upper part of the tunnel, results suggest that A2062 and A2451 can communicate in both directions for translation stalling, mostly through dynamically coupled C2063, C2064, and A2450.

My comment:  This is truly awe-inspiring. The functional group A2451, which is not only of crucial importance as described above for peptyde bond catalysis, but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here" !! and the ribosome takes action. 

Prebiotic protein folding is indeed a huge problem, since we know now that the ribosome has a helping hand in promoting the right folding. And when that does not occur, no deal. So you need a ribosome to have functional protein folds, but you need functional folds to make a ribosome. What came first ?

If all this is not evidence of a bioengineered process, i don't know, what is!!



1. https://www.biorxiv.org/content/10.1101/2020.09.01.277582v1.full

Amazing surveillance pathways that rescue ribosomes lost in translation point to intelligently designed mechanisms

https://reasonandscience.catsboard.com/t2984-error-check-and-repair-during-messenger-rna-translation-in-the-ribosome-by-chance-or-design#8048

The final step of the central dogma is the most complex, where the information in RNA is translated to build proteins in the ribosome.  Ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for Instance, for a messenger RNA that has broken and is thus is missing its stop codon. The ribosome uses an incredible peace of bio-engineering to rescue a stalled situation when they get stuck at the end of the truncated chain. They use a mechanism called Trans-translation. It is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. This mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frame shifting. Trans-Translation is performed by a ribonucleoprotein complex. A strangely shaped RNA molecule mimics both a transfer RNA and a messenger RNA, restarting the process and cleaning up the mess. A proteolysis-inducing tag is added at the unfinished polypeptide, and facilitates the degradation of the aberrant messenger RNA. Trans-translation relies on two main factors: small stable 10S RNA (ssrA), an aminoacylated transfer-messenger RNA (tmRNA) with properties both of a tRNA and an mRNA; and SmpB (small protein B). 

My comment: This is awesome. Wow!! How could random non-guided, non-intelligent mechanisms have foresight, and the knowledge of this problem of truncated messenger RNA's that do not have a stop codon, and a sophisticated mechanism to deal with it? This is a masterfully crafted salvage mechanism in order to prevent the cell to produce toxic polymer strands that would accumulate, and in the end, destroy the cell. This is an engineering marvel of extraordinary sophistication.


Transfer-messenger RNA.
Transfer-messenger RNA (top ) includes a portion that mimics a transfer RNA ( red ) and a portion that mimics a messenger RNA ( magenta ), complete with a stop codon. It binds to stalled ribosomes ( bottom ), resuming synthesis using its own short message. Amazingly, this message encodes a small tag that is added to the end of the truncated protein, signaling to the cell that the protein is faulty and needs to be destroyed

Transfer-messenger RNA
https://en.wikipedia.org/wiki/Transfer-messenger_RNA

Transfer-messenger RNA (abbreviated tmRNA ) is a bacterial RNA molecule with dual tRNA-like and messenger RNA-like properties. The tmRNA forms a ribonucleoprotein complex (tmRNP) together with 
- Small Protein B (SmpB), 
- Elongation Factor Tu (EF-Tu), and 
- ribosomal protein S1. 
In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein biosynthesis, for example when reaching the end of a messenger RNA which has lost its stop codon. The tmRNA is remarkably versatile: it recycles the stalled ribosome, adds a proteolysis-inducing tag to the unfinished polypeptide, and facilitates the degradation of the aberrant messenger RNA

Biology of trans-Translation
The trans-translation mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. trans-Translation is performed by a ribonucleoprotein complex composed of tmRNA, a specialized RNA with properties of both a tRNA and an mRNA, and the small protein SmpB. tmRNA-SmpB interacts with translational complexes stalled at the 3prime end of anmRNA to release the stalled ribosomes and target the nascent polypeptides and mRNAs for degradation. In addition to quality control pathways, some genetic regulatory circuits use transtranslation to control gene expression. Diverse bacteria require transtranslation when they execute large changes in their genetic programs, including responding to stress, pathogenesis, and differentiation. 1

Genes encoding tmRNA and SmpB are present throughout the bacterial kingdom.


trans-Translation removes all components of stalled translation complexes. 
tmRNA binds to SmpB and is aminoacylated by alanyl-tRNA synthetase (AlaRS). EF-Tu in the GTP state binds to alanyl-tmRNA, activating the complex for ribosome interaction. The alanyl-tmRNA/SmpB/EF-Tu complex recognizes ribosomes at the 3' end of an mRNA and enters the A-site as though it were a tRNA. The nascent polypeptide is transferred to tmRNA, and the tmRNA tag reading frame replaces the mRNA in the decoding center. The mRNA is rapidly degraded. Translation resumes, using tmRNA as a message, resulting in addition of the tmRNA-encoded peptide tag to the C terminus of the nascent polypeptide. Translation terminates at a stop codon in tmRNA, releasing the ribosomal subunits and the tagged protein. Multiple proteases recognize the tmRNA tag sequence and rapidly degrade the protein (box 3).

1. https://sci-hub.st/https://www.annualreviews.org/doi/10.1146/annurev.micro.62.081307.162948
2. https://sci-hub.st/https://www.nature.com/articles/nrm3457?proof=t

How The Folded Structure (And Then The “Loading”) Of tRNA Corrects Attempts To Reduce Protein Synthesis To “Mere” Chemistry

The mRNA tape, of course, carries initiation, stepwise elongation, finite succession and halting. Thus we see an algorithm at work, with an information bearing tape as controller and an assembly line using mobile position-arm units.

This is seriously advanced automation and manufacturing, using algorithms and code on string data structures [let’s only mention editing to form mRNAs], thus language. Where the coding is present twice in two separate subsystems, processed effectively independent of one another. DNA is unzipped and used to assemble mRNA precursors, which are edited to form the control tape. Separately, tRNA is created with the implicit anticodon, again stored in the master tape, DNA. Properly folded tRNAs are loaded with the appropriate AA or a precursor that is modified to be correct.

These are then brought into the ribosome under proper manufacturing control and peptide chains are built for further formation into proteins used in cellular processes including this one.

Chicken-egg loops abound, pointing to Functionally Specific, Complex Organisation and/or associated Information (FSCO/I)FSCO/I and islands of function requiring initial manufacture to a design, on pain of fruitlessly, aimlessly wandering in seas of non function and exhausting the blind search capability of the observed cosmos.

At this stage, it is manifest why a design inference on protein synthesis is robust and empirically grounded. As for means:

clever designer[s] + molecular nanotech lab –> clever FSCO/I rich design

Of course, one is free to reject such, but in all responsible fairness needs to provide a cogent, empirically warranted explanation.>>

Such an empirically warranted, blind watchmaker blind chance and/or mechanical necessity explanation remains conspicuously absent, even as agent explanations are ideologically locked out, never mind the significance of LANGUAGE and ALGORITHMS here.

Functional, Specific, Complex Organization and/or associated Information (FSCO/I)FSCO/I points to design
1. The ribosome is an extremely advanced automation and manufacturing unit,  where algorithms and code on string data structure, thus language, is processed. Coding starts in two separate subsystems, processed effectively independent of one another. DNA is unzipped and used to assemble mRNA precursors, which are edited to form the control tape. Separately, tRNA is created with the implicit anticodon, again stored in the master tape, DNA. Once mRNA enters the Ribosome, codons dictate the correct loading of tRNA's with the appropriate amino acids. These are then brought into the ribosome under proper manufacturing control and peptide chains are built for further formation into proteins used in cellular processes.
2. There is a Functional, Specific, Complex Organization and/or associated Information (FSCO/I)FSCO/I  requiring initial manufacture to a design, on pain of fruitlessly, aimlessly wandering in seas of non function and exhausting the blind search capability of the observed cosmos. Chicken-egg loops abound.
3. Of course, one is free to reject an intelligent design hypothesis, but in all responsible fairness needs to provide an alternative cogent, sound & warranted explanation. Such a  warranted, blind watchmaker blind chance and/or mechanical necessity explanation remains conspicuously absent, when agent explanations are ideologically locked out.
At this stage, it is manifest why a design inference on protein synthesis is robust and empirically grounded. As for means: Clever designer[s] + molecular nanotech lab –> clever FSCO/I rich design



So, now, we can see the molecular nanotech in action and how it uses a base of Chemical-Physical processes, but we actually have a layer-cake architecture, once code enters as codes are based on arbitrary symbols that have to be decoded on the receiver side of a system. Echoing Yockey as seen above, the general architecture:



It is noteworthy that algorithmic, alphanumeric code — a linguistic phenomenon — remains stubbornly as only the product of intelligence.

https://uncommondescent.com/design-inference/how-the-folded-structure-and-then-the-loading-of-trna-corrects-attempts-to-reduce-protein-synthesis-to-mere-chemistry/#comment-701278

The Ribosome is irreducibly complex

https://reasonandscience.catsboard.com/t1661p25-translation-through-ribosomes-amazing-nano-machines#8340

Eugene Koonin, the logic of chance:
Comparative genomic reconstructions of the gene repertoire of LUCA(S) point to a complex translation system that includes at least 18 of the 20 aminoacyl-tRNA synthetases (aaRS), several translation factors, at least 40 ribosomal proteins, and several enzymes involved in rRNA and tRNA modification. It appears that the core of the translation system was already fully shaped in LUCA(S) (Anantharaman, et al., 2002).

Ribosomes consist of two different sized subunits. Ribosomal proteins dominate these subunit structures, but there are up to 120 different molecules involved: rRNA, mRNAs, tRNAs, ribosomal proteins, aminoacyl-synthetases, and scanning factors. They are all needed to fulfil this basic, yet highly complex cellular housekeeping function. 1

The Ribosome
1. Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. They form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.
2. The set up of a language, and upon it, the programming of a completely autonomous communication network, which directs the operation of a complex factory, which during operation error checks and performs repairs, to make specific purposeful products, is always the product of an intelligent agency.
3. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confesses that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: an intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

Evidence of design in the ribosome
1. Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. For example, the functional group A2451, one single ribonucleotide which is not only of vital importance for peptide bond catalysis ( yeah, how did unguided events "discover" hot to place this one tiny molecule in the right place amongst 2900 of its peers ) but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here" !! and the ribosome takes action. Ribosomes form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, they can halt the translation process on the fly, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential. Even more strikingly amazing, ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for instance, for a messenger RNA that has broken and is thus is missing its stop codon. The ribosome uses an incredible piece of bio-engineering to rescue a stalled situation when they get stuck at the end of the truncated chain. They use a mechanism called Trans-translation. It is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. This mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. This so-called Trans-Translation is performed by a ribonucleoprotein complex.
2. The set up of a language, and upon it, the programming of a completely autonomous communication network, which directs the operation of a complex factory, which during operation error checks and performs repairs, to make specific purposeful products, is always the product of an intelligent agency. 
3. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confesses that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: an intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

Evidence of design in the ribosome
1. Ribosomes have the purpose to translate genetic information into proteins. According to Craig Venter, the ribosome is “an incredibly beautiful complex entity” which requires a minimum of 53 proteins. It is nothing if not an editorial perfectionist…the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products…  They are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They can halt the translation process on the fly, and coordinate extremely complex movements. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.
2. For the assembly of this protein-making factory, consisting of multiple parts, the following is required: genetic information to produce the ribosome assembly proteins, chaperones, all ribosome subunits, and assembly cofactors. a full set of tRNA's, a full set of aminoacyl tRNA synthetases, the signal recognition particle, elongation factors, mRNA, etc. The individual parts must be available,  precisely fit together, and assembly must be coordinated. A ribosome cannot perform its function unless all subparts are fully set up and interlocked. 
3. The making of a translation machine makes only sense if there is a source code, and information to be translated. Eugene Koonin: Breaking the evolution of the translation system into incremental steps, each associated with a biologically plausible selective advantage is extremely difficult even within a speculative scheme let alone experimentally. Speaking of ribosomes, they are so well structured that when broken down into their component parts by chemical catalysts (into long molecular fragments and more than fifty different proteins) they reform into a functioning ribosome as soon as the divisive chemical forces have been removed, independent of any enzymes or assembly machinery – and carry on working.  Design some machinery which behaves like this and I personally will build a temple to your name! Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. The origin of the ribosome is better explained through a brilliant intelligent and powerful designer, rather than mindless natural processes by chance, or/and evolution since we observe all the time minds capabilities producing machines and factories.

Translation through the ribosome is an irreducible, integrated complex process
1. The ribosome is the 3D printer of proteins. A human-made 3D printer is made of several functional parts, like the nozzle, the extruder, cooling fan, heated be, the painter's tape, etc. The 3D extruder has no use on its own. But only, when working inside the 3D printer in the right place. A bacterial cell depends upon a translation and coding system consisting of 106 distinct but functionally integrated proteins as well several distinct types of RNA molecules (tRNAs, mRNAs, and rRNAs). This system includes the ribosome (consisting of fifty distinct protein parts), the twenty distinct tRNA synthetases, twenty distinct tRNA molecules with their specific anticodons, about 200 ribosome assembly proteins and 75 co-factors, chaperones, free-floating amino acids, ATP molecules (for energy), and—last, but not least—information-rich mRNA transcripts for directing protein synthesis. Many of the proteins in the translation system perform multiple functions and catalyze coordinated multistep chemical transformations.
2. In the same sense, as an engineer would not project, invent, create and make a blueprint of a 3D printer extruder with no use by its own, but only conjoined, and together with all other parts while projecting a whole printer, envisioning its end function and use, its evident that unguided random natural events without foresight would not come up with an assemblage of tiny molecular machines, enzymatic structures with unique contours, which bear no function by their own, but only when inserted as part of the ribosome with higher ends, being essential for cells to translate DNA information into proteins, and being a key part participating to perpetuate life. Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. In other words: Why would natural selection select an intermediate biosynthesis product, which has by its own no use for the organism, unless that product keeps going through all necessary steps, up to the point to be ready to be assembled in a larger system?  Never do we see blind, unguided processes leading to complex functional systems with integrated parts contributing to the overarching design goal. A minimal amount of instructional complex information is required for a gene to produce useful proteins. A minimal size of a protein is necessary for it to be functional.   Thus, before a region of DNA contains the requisite information to make useful proteins, natural selection would not select for a positive trait and play no role in guiding its evolution.
3. Naturalistic mechanisms or undirected causes do not suffice to explain the origin of information (instructed complex information), irreducible complexity, and the setup of complex machines with little tolerance of change.   Therefore, intelligent design constitutes the best explanations for the origin of the information guiding the making of the irreducible and integrated complex ribosome protein factory.


Evidence of design in the ribosome
1.  Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. For example, the functional group A2451, one single ribonucleotide which is not only of vital importance for peptide bond catalysis ( yeah, how did unguided events "discover" hot to place this one tiny molecule in the right place amongst 2900 of its peers ) but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here" !! and the ribosome takes action. Ribosomes form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, they can halt the translation process on the fly, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential. Even more strikingly, ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for instance, for a messenger RNA that has broken and is thus is missing its stop codon. The ribosome uses an incredible piece of bio-engineering to rescue a stalled situation when they get stuck at the end of the truncated chain. They use a mechanism called Trans-translation. It is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. This mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. This so-called Trans-Translation is performed by a ribonucleoprotein complex.
2. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confesses that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: 
3. An intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

The adapter key machinery to translate genetic Information is irreducibly complex
1. Ribosomes have the purpose to translate genetic information into proteins. According to Craig Venter, the ribosome is “an incredibly beautiful complex entity” which requires a minimum of 53 proteins. It is nothing if not an editorial perfectionist…the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products…  They are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. Furthermore, they communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They can halt the translation process on the fly, and coordinate extremely complex movements. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.
2. For the assembly of this protein making factory, consisting of multiple parts, the following is required: genetic information to produce the ribosome assembly proteins, chaperones, all ribosome subunits and assembly cofactors. A full set of tRNA's, a full set of aminoacyl tRNA synthetases, the signal recognition particle, elongation factors, mRNA, etc. The individual parts must be available,  precisely fit together, and assembly must be coordinated. A ribosome cannot perform its function unless all subparts are fully set up and interlocked.
3. The making of a translation machine makes only sense if there is a source code, and information to be translated. Eugene Koonin: Breaking the evolution of the translation system into incremental steps, each associated with a biologically plausible selective advantage is extremely difficult even within a speculative scheme let alone experimentally. Speaking of ribosomes, they are so well-structured that when broken down into their component parts by chemical catalysts (into long molecular fragments and more than fifty different proteins) they reform into a functioning ribosome as soon as the divisive chemical forces have been removed, independent of any enzymes or assembly machinery – and carry on working.  Design some machinery which behaves like this, and I personally will build a temple to your name! Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. The origin of the ribosome is better explained through a brilliant intelligent and powerful designer, rather than mindless natural processes by chance, or/and evolution since we observe all the time minds capabilities producing machines and factories.


The following are the essential parts to a) assemble, and b) operate the translation machinery of genetic information in the cell:

Prebiotic Evolution and Astrobiology, J. Tze-Fei Wong, PhD Antonio Lazcano, PhD 2009 Landes Bioscience

Translation, ribosomal structure and biogenesis
8 Glutamyl-tRNA synthetase
9 Putative translation factor (SUA5)
12 Predicted GTPase, probable translation factor
13 Alanyl-tRNA synthetase
16 Phenylalanyl-tRNA synthetase alpha subunit
17 Aspartyl-tRNA synthetase
18 Arginyl-tRNA synthetase
23 Translation initiation factor 1 (eIF-1/SUI1) and related proteins
24 Methionine aminopeptidase
30 Dimethyladenosine transferase (rRNA methylation)
48 Ribosomal protein S12
49 Ribosomal protein S7
51 Ribosomal protein S10
52 Ribosomal protein S2
60 Isoleucyl-tRNA synthetase
72 Phenylalanyl-tRNA synthetase beta subunit
80 Ribosomal protein L11
81 Ribosomal protein L1
87 Ribosomal protein L3
88 Ribosomal protein L4
89 Ribosomal protein L23
90 Ribosomal protein L2
91 Ribosomal protein L22
92 Ribosomal protein S3
93 Ribosomal protein L14
94 Ribosomal protein L5
96 Ribosomal protein S8
97 Ribosomal protein L6P/L9E
98 Ribosomal protein S5
99 Ribosomal protein S13
100 Ribosomal protein S11
101 Pseudouridylate synthase
102 Ribosomal protein L13
103 Ribosomal protein S9
124 Histidyl-tRNA synthetase
130 Pseudouridine synthase
143 Methionyl-tRNA synthetase
162 Tyrosyl-tRNA synthetase
172 Seryl-tRNA synthetase
180 Tryptophanyl-tRNA synthetase
182 Predicted translation initiation factor 2B subunit, eIF-2B α/β/δ family
184 Ribosomal protein S15P/S13E
185 Ribosomal protein S19
186 Ribosomal protein S17
197 Ribosomal protein L16/L10E
198 Ribosomal protein L24
199 Ribosomal protein S14
200 Ribosomal protein L15
231 Translation elongation factor P (EF-P)/translation initiation factor 5A
244 Ribosomal protein L10
252 L-asparaginase/archaeal Glu-tRNAGln amidotransferase subunit D
255 Ribosomal protein L29
256 Ribosomal protein L18
343 Queuine/archaeosine tRNA-ribosyltransferase
361 Translation initiation factor 1 (IF-1)
423 Glycyl-tRNA synthetase (class II)
441 Threonyl-tRNA synthetase
442 Prolyl-tRNA synthetase
480 Translation elongation factors (GTPases)
495 Leucyl-tRNA synthetase
522 Ribosomal protein S4 and related proteins
525 Valyl-tRNA synthetase
532 Translation initiation factor 2 (IF-2; GTPase)
621 2-Methylthioadenine synthetase
1093 Translation initiation factor 2, alpha subunit (eIF-2alpha)
1258 Predicted pseudouridylate synthase
1325 Predicted exosome subunit
1358 Ribosomal protein HS6-type (S12/L30/L7a)
1369 RNase P/RNase MRP subunit POP5
1383 Ribosomal protein S17E
1384 Lysyl-tRNA synthetase (class I)
1471 Ribosomal protein S4E
1491 Predicted RNA-binding protein
1498 Protein implicated in ribosomal biogenesis Nop56p homolog
1499 NMD protein affecting ribosome stability and mRNA decay
1500 Predicted exosome subunit
1503 Peptide chain release factor 1 (eRF1)
1514 2ʹ-5ʹ RNA ligase
1534 Predicted RNA-binding protein containing KH domain possibly ribosomal protein
1549 Queuine tRNA-ribosyltransferases, contain PUA domain
1552 Ribosomal protein L40E
1588 RNase P/RNase MRP subunit p29
1601 Translation initiation factor 2, beta subunit (eIF-2beta)/eIF-5 N-terminal domain
1603 RNase P/RNase MRP subunit p30
1631 Ribosomal protein L44E
1632 Ribosomal protein L15E
1676 tRNA splicing endonuclease
1717 Ribosomal protein L32E
1727 Ribosomal protein L18E
1736 Diphthamide synthase subunit DPH2
1746 tRNA nucleotidyltransferase (CCA-adding enzyme)
1798 Diphthamide biosynthesis methyltransferase
1841 Ribosomal protein L30/L7E
1867 Dimethylguanosine tRNA methyltransferase
1889 Fibrillarin-like rRNA methylase
1890 Ribosomal protein S3AE
1911 Ribosomal protein L30E
1976 Translation initiation factor 6 (eIF-6)
1997 Ribosomal protein L37AE/L43A
1998 Ribosomal protein S27AE
2004 Ribosomal protein S24E
2007 Ribosomal protein S8E
2016 Predicted RNA-binding protein (contains PUA domain)
2023 RNase P subunit RPR2
2051 Ribosomal protein S27E
2053 Ribosomal protein S28E/S33
2058 Ribosomal protein L12E/L44/L45/RPP1/RPP2
2075 Ribosomal protein L24E
2092 Translation elongation factor EF-1beta
2097 Ribosomal protein L31E
2125 Ribosomal protein S6E (S10)
2126 Ribosomal protein L37E
2139 Ribosomal protein L21E
2147 Ribosomal protein L19E
2157 Ribosomal protein L20A (L18A)
2163 Ribosomal protein L14E/L6E/L27E
2167 Ribosomal protein L39E
2174 Ribosomal protein L34E
2238 Ribosomal protein S19E (S16A)
2260 Predicted Zn-ribbon RNA-binding protein
2263 Predicted RNA methylase
2511 Archaeal Glu-tRNAGln amidotransferase subunit E (contains GAD domain)
2519 tRNA(1-methyladenosine) methyltransferase and related methyltransferases
2888 Predicted Zn-ribbon RNA-binding protein with a function in translation
2890 Methylase of polypeptide chain release factors
3277 RNA-binding protein involved in rRNA processing
5256 Translation elongation factor EF-1alpha (GTPase)
5257 Translation initiation factor 2, gamma subunit (eIF-2gamma; GTPase)

Translation:
Irreducible parts:
mRNA
genetic code
45 tRNAs
20 different types of amino acids
20 different types of aminoacyl-tRNA-synthetases  aaRSs are essential components for protein synthesis in every living species
proofreading function
prokaryotic ribosomes
small subunit (designated 30S): 16S rRNA and 21 proteins
The large subunit (50S): 23S and 5S rRNAs and 34 proteins
eukaryotic ribosomes
small subunit (40S):18S rRNA and 30 proteins
large subunit (60S): 28S, 5.8S, and 5S rRNAs and about 45 proteins
AUG codon to start translation  ( GUG codon in some bacteria)

Irreducible stages:
Initiation, elongation, and termination

Initiation: the first step of the initiation stage is the binding of a specific initiator methionyl tRNA and the mRNA to the small ribosomal subunit. The large ribosomal subunit then joins the complex, forming a functional 1.ribosome on which elongation of the polypeptide chain proceeds.
Aminoacylated and formylated initiator tRNA (fMet-tRNAfMet)

A number of specific non-ribosomal proteins are also required for the various stages of the translation process:
Prokaryotes: IF-1, IF-2, IF-3
Eukaryotes: eIF-1, eIF-1A, eIF-2, eIF-2B, eIF-3, eIF-4A, eIF-4B, eIF-4E, eIF-4G, eIF-5
Elongation
Prokaryotes:  EF-Tu, EF-Ts, EF-G
Eukaryotes:   eEF-1α, eEF-1βγ, eEF-2
Elongation factor (EF-Tu in prokaryotes, eEF-1α in eukaryotes)
release factors (RF-1, RF-2 and RF-3 in prokaryotes, in eukaryotic cells release factors (eRF-1,and eRF-3)
Termination
Prokaryotes: RF-1, RF-2, RF-3
Eukaryotes: eRF-1, eRF-3

Biogenesis of the ribosome: 
The Ribosome requires over 200 scaffold proteins, chaperones, and 75 cofactors for its assembly.
maturation of tRNAs:
Proteins:
CCA tRNA nucleotidyltransferase 1,
Zinc phosphodiesterase ELAC protein 2
and  Enzymatic  complexes:
Ribonuclease P: Human nuclear RNase P consists of 10 Protein subunits and one RNA subunit.
tRNA ligase complex
tRNA-splicing endonuclease
All three RNA polymerases,
Numerous small nucleolar RNAs (snoRNAs)
Several hundred protein assembly factors AF's 2
The Tor signaling pathway controls ribosome biogenesis at different levels.
In eukaryotic cells: 
>350 nonribosomal factors
The trans-translation mechanism which is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors.
The splicing apparatus
C/D box snoRNAs (SNORDs)
H/ACA snoRNPs
TRAMP4/5 complex ( necessary for degradation of kinetically dead-end products )
nucleolin (NCL)
nucleophosmin (NPM)
protein 56 (NOP56)
PeBoW complex
dyskerin (DKC)



Steps in Translation

Components necessary for the initiation of translation include the interaction of the small and large ribosomal subunits, mRNA, an initiator aminoacyl-tRNA, GTP, and a large group of initiation factors. The initiation complex is formed by the small ribosomal subunit, the mRNA and Met-tRNA (methionine attached to a specialized initiator tRNA type). The Met-tRNA is hydrogen bonded to the AUG initiation codon on the mRNA
Formation of the ribosomal initiation complex is completed with the addition of the large ribosomal subunit. The AUG codon and the Met-tRNA are positioned in the P (peptidyl) site of the ribosome.
New aminoacyl-tRNA is then positioned in the A (aminoacyl) site.
The covalent bond between the amino acid and the tRNA in the P site is broken.
A peptide (covalent) bond is formed betwteen the two amino acids.
The empty tRNA then dissociates from the P site.
Translocation of the ribosome occurs such that the peptidyl-tRNA in the A site is translocated into the P site.
The complex now looks very similar to that at the initiation of translation. The peptidyl-tRNA is in the P site and the A site is empty and ready to accept the next aminoacyl-tRNA.
The amino acid in the P site is separated from its tRNA and peptide bond formation takes place with the aminoacyl-tRNA in the A site. The tRNA is liberated from the P site and the ribosome tranlocates such that the new peptidyl-tRNA is in the P site; the A site is ready to accept the next aminoacyl-tRNA.
The elongation cycle (the addition of amino acids one at a time to a growing polypeptide chain) continues for more peptide bond formation and translocation.
A stop codon (UAA, UAG, or UGA) positions in the A site; a stop codon has no anticodon (an aminoacyl tRNA that will hydrogen bond to this codon); for this reason, no other amino acid will be added.
The stop codon is reconized and bound by a protein called the termination or release factor.
The release factor binds to the stop codon at the A site and begins a sequence of events that brings about the termination of translation.
The termination sequence begin with the dissociation of the newly synthesized protein from the peptidyl-tRNA in the P site.
Separation of the newly synthesized proteins from the ribosome is followed by the dissocation of all the remaining subunits.
The ribosomal subunits and the mRNA can reassemble with Met-tRNA to form new initation complexes and prteins translation can begin again to produce additional copies of the protein.

Initiation
A ribosome separates into large and small subunits
Met-tRNA combines with GTP in a side reaction involving an initiation factor
Met-tRNA is added to the small ribosomal subunit
The small subunit is added to the mRNA in a reaction driven by ATP hydrolysis; attachment takes place at the 5' cap of the mRNA; once attached, tha small subunits moves or "scans" along the mRNA until it reaches the AUG initiator codon
The large ribosomal subunit is added driven by the hydrolysis of GTP brought to the complex with the initiator tRNA; elongation follows

Elongation
Aminoacyl-tRNA binds to the A site
Peptide bond formation
Peptidyl-tRNA formed at the A site by step two is transferred from the A site to the P site

Peptide Bond Formation
Peptide bond formation is catalyzed on the ribosome by peptidyl transferase. (a) Adjacent aminoacyl-tRNAs bound to the mRNA at the ribosome (b) folowing peptide bond formation, an uncharged tRNA is in the P site and a dipeptidyl-tRNA in the A site.

Termination of Translation
The ribosome recognizes a chain termination codon (here, UAG) with the aid of release factors. The release factor reads the stop codon, and this initiatiartes a series of specific termination events leading to the release of the completed polypeptide
Stop codon is encountered at the A site which causes the release factor to bind to the A site along with GTP insead of aminoacyl-tRNA
The release factor binds to the stop codon and hydrolysis of the bond holding the polypeptide chain to the tRNA site at the P site, catalyzed by the peptidyl tranferase site of the large subunit
Since there is no amino acid located at the A site, the hydrolysis allows the polypeptide chain to be freed from the ribosome; with the release of the polypeptide, the release factor is ejected from the A site, and the empty tRNA is ejected from the P site
Ribosomal components separate





The Ribosome is irreducibly complex

The foregoing discussion leads us to conclude that the ribosome in itself is irreducibly complex, requiring several dozen proteins to be present at the same time in order for it to work. Furthermore, the molecular machinery that regulates ribosomal gene co-expression involves just under 300 transcription regulators, which is also further modulated according to several cell types in humans and mice.



1. https://creation.com/ribosomes-and-design
2. https://www.bzh.db-engine.de/group/56/martin%20koš/setLang=en

https://www.evolutionisamyth.com/biological/ribosomes-irreducibly-complex-ingeniously-designed/

Ribosomes Optimized for Speed, Flexibility

The DNA translation machines in the cell show unexpected complexity, forcing molecular biologists to revise what they thought they knew about ribosomes. In particular, they appear optimized for speed of self-duplication and modularized for flexibility.

Last September, we evaluated a fascinating paper about ribosomes that showed how this molecular machine that translates DNA “requires the orchestrated function of hundreds of proteins” — and that’s just to get to the “pre-ribosome” stage! Ribosomes are marvels of organization and function. Since then, more discoveries have shown additional design features of ribosomes. Before the cell can divide, all the proteins needed by the two daughter cells must be translated. This requirement effectively doubles the work for these machines. How does the cell prepare for this increased workload? Rather than speed up translation, the ribosomes first duplicate themselves, effectively doubling the production capacity. This means that they have to prepare and assemble all their own RNAs and proteins first. Without efficient ways to accomplish this prerequisite, cell division could be seriously delayed.

An interesting model, published in Nature by Johan Paulsson’s team at Harvard, suggests that “Ribosomes are optimized for autocatalytic production.” They knew that ribosomes are already optimized in three ways. Now, they add a fourth:

Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes — such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55–80 small, similarly sized segments — speed up their autocatalytic production. However, in recent years it has also become clear that ribosomes are exceptional as products of the ribosomal machinery. Not only do ribosomal proteins (r-proteins) make up a large fraction of the total protein content in many cells, but the autocatalytic nature of ribosome production introduces additional constraints. Specifically, the ribosome doubling time places a hard bound on the cell doubling time, because for every additional ribosome to share the translation burden there is also one more to make. Even for the smallest and fastest ribosomes, it takes at least 6 min, and typically much longer, for one ribosome to make a new set of r-proteins (Supplementary Information); and this estimate does not account for the substantial time that is invested in the synthesis of ternary complexes. This bound seems to explain the observed limits on bacterial growth, because ribosomes must also spend much of their time making other proteins

Structures of the human and Drosophila 80S ribosome
In contrast to their bacterial counterparts, eukaryotic ribosomes are much larger and more complex; they contain approximately 2,650 nucleotides of additional rRNA in H. sapiens in the form of so-called expansion segments and 26 additional ribosomal proteins as well as 2,452 amino acids of ribosomal protein extensions. 1



Layers of the eukaryotic ribosome. 
a–f, Surface representations (a, c, e) and schematics (b, d, f) of the bacterial T. thermophilus 70S ribosome (a, b) 47, the S. cerevisiae 80S ribosome (c, d) 10 (the eukaryote-specific protein–RNA layer is shown), and the mammalian 80S ribosome from H. sapiens (e, f) (the two additional layers, RNA–RNA and RNA-only, are shown). SB, P-stalk base; Sp, spur; TE, tunnel exit.

The majority of the rRNA and ribosomal proteins that constitute the bacterial 70S ribosome is conserved in eukaryotes, and can therefore be considered to form the core of the 80S ribosome (Fig. a, b). Structures of the yeast and Tetrahymena ribosomes have revealed that the additional eukaryote-specific ribosomal proteins form a network of interactions with the rRNA expansion segments, resulting in an intertwined RNA–protein layer6–10 (Fig. c, d). In metazoan eukaryotes, this RNA–protein layer has increased in size and complexity owing to the presence of additional ribosomal protein extensions and rRNA expansion-segment insertions (Fig. e, f ). Moreover, the substantial increase in RNA mass of metazoans, particularly mammalian ribosomes, compared to yeast and protists, has resulted in the presence of two additional RNA layers (Fig. e, f): a rigid inner layer, resulting from multiple RNA–RNA tertiary interactions, followed by aflexible outer layer, arisingfrom helical insertions and extensions of the rRNA expansion segments. 

The Structure and Function of the Eukaryotic Ribosome
However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. 2


The bacterial and eukaryotic small ribosomal subunit.
(A,B) Interface (upper) and solvent (lower) views of the bacterial 30S subunit (Jenner et al. 2010a). (A) 16S rRNA domains and associated r-proteins colored distinctly: b, body (blue); h, head (red); pt, platform (green); and h44, helix 44 (yellow). (B) 16S rRNA colored gray and r-proteins colored distinctly and labeled. (C–E) Interface and solvent views of the eukaryotic 40S subunit (Rabl et al. 2011), with (C) eukaryotic-specific r-proteins (red) and rRNA (pink) shown relative to conserved rRNA (gray) and r-proteins (blue), and with (D,E) 18S rRNA colored gray and r-proteins colored distinctly and labeled.


The bacterial and eukaryotic large ribosomal subunit.
(A) Interface (upper) and solvent (lower) views of the bacterial 50S subunit (Jenner et al. 2010b), with 23S rRNA domains and bacterial-specific (light blue) and conserved (blue) r-proteins colored distinctly: cp, central protuberance; L1, L1 stalk; and St, L7/L12 stalk (or P-stalk in archeaa/eukaryotes). (B–E) Interface and solvent views of the eukaryotic 60S subunit (Klinge et al. 2011), with (B) eukaryotic-specific r-proteins (red) and rRNA (pink) shown relative to conserved rRNA (gray) and r-proteins (blue), (C) eukaryotic-specific expansion segments (ES) colored distinctly, and (D,E) 28S rRNA colored gray and r-proteins colored distinctly and labeled.




1. https://sci-hub.ren/10.1038/nature12104
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3331703/