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Defending the Christian Worlview, Creationism, and Intelligent Design

This is my personal virtual library, where i collect information, which leads in my view to the Christian faith, creationism, and Intelligent Design as the best explanation of the origin of the physical Universe, life, and biodiversity


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Defending the Christian Worlview, Creationism, and Intelligent Design » Origin of life » Translation through ribosomes, amazing nano machines

Translation through ribosomes, amazing nano machines

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Otangelo


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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.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1082788/

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:  Keeping a stable internal cellular environment requires constant adjustments as conditions change inside and outside the cell. The adjusting of systems within a cell is called homeostatic regulation. Because the internal and external environments of a cell are constantly changing, adjustments must be made continuously to stay at or near the set point (the normal level or range).
Regulation is ubiquitous in biology. Regulation of biological processes occurs when any process is modulated in its frequency, rate or extent. Biological processes are regulated by many means; examples include the control of gene expression, protein modification or interaction with a protein or substrate molecule. While Gene expression in prokaryotes is regulated primarily at the transcriptional level, in eukaryotic cells, it is regulated at many levels (epigenetic, transcriptional, post-transcriptional, translational, and post-translational). Metabolism or intracellular signaling also requires regulation. Gene regulation is a dynamical process composed of a number of steps, for example the binding of Transcription Factors to DNA, recruitment of transcription machinery and the production of the messenger RNA, post-transcriptional regulation, splicing and transport of mRNA, translation, maturation and possible localization of proteins. Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; according to one estimate, 70% of genes in humans are expressed as multiple proteins through alternative splicing. The primary method to control what type and how much protein is expressed in a prokaryotic cell is through the regulation of DNA transcription into RNA.

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

Properties of Life
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/

Regulate means control or maintain the rate or speed of (a machine or process) so that it operates properly. Regulation is the management of complex systems according to a set of rules and trends. It is a rule or directive made and maintained by an authority.
Regulating is always either a) a preprogrammed action by an intelligent agency, or b) an action where an intelligent agent is directly actively involved. Setting rules, giving directives for specific purposes or functions is always the result of a mental action. It is logical to conclude that biological regulation requires intelligent set up.


Regulation is essential in biology
1. Regulation is ubiquitous in biology, and a fundamental property of living systems. Keeping a stable internal cellular environment requires constant adjustments as conditions change inside and outside the cell. The adjusting of systems within a cell is called homeostatic regulation. Because the internal and external environments of a cell are constantly changing, adjustments must be made continuously to stay at or near the set point (the normal level or range). Regulation of biological processes occurs when any process is modulated in its frequency, rate or extent. Biological processes are regulated by many means; examples include the control of gene expression, protein modification or interaction with a protein or substrate molecule. While Gene expression in prokaryotes is regulated primarily at the transcriptional level, in eukaryotic cells, it is regulated at many levels (epigenetic, transcriptional, post-transcriptional, translational, and post-translational). Metabolism or intracellular signaling also requires regulation. Gene regulation is a dynamical process composed of a number of steps, for example the binding of Transcription Factors to DNA, recruitment of transcription machinery and the production of the messenger RNA, post-transcriptional regulation, splicing and transport of mRNA, translation, maturation and possible localization of proteins. 
2. 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. Regulate means control or maintain the rate or speed of (a machine or process) so that it operates properly. Regulation is the management of complex systems according to a set of rules and trends. It is a rule or directive made and maintained by an authority. Regulating is always either a) a preprogrammed action by an intelligent agency, or b) an action where an intelligent agent is directly actively involved. 
3. Setting rules, giving directives for specific purposes or functions is always the result of a mental action. It is logical to conclude that biological regulation requires intelligent set up.

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 . Here we focus on translation initiation in bacteria.  Archaeal and eukaryotic processes of translation initiation are reviewed elsewhere.

BACTERIAL TRANSLATION INITIATION
Ribosomes initiate translation on mRNAs already during transcription. Hence, transcription and translation are tightly coupled cellular processes. Translation initiation is the rate-limiting and most highly regulated phase of the four phases in protein biosynthesis.

The rate at which ribosomes assemble on the mRNA is on the order of seconds, although it is specific for each mRNA. The ribosomes subsequently translate the mRNA at a rate of approximately 12 amino acids per second . The ribosome, the aminoacylated and formylated initiator tRNA (fMet-tRNAfMet), mRNA, and the three protein factors, initiation factor IF1, initiation factor IF2, and initiation factor IF3, are involved in the translation initiation phase

Translation through ribosomes,  amazing nano machines - Page 2 Transl11
Translation initiation pathway in bacteria.
The 30S and 50S ribosomal subunits are shown in light and dark grey, respectively. Translation initiation factors IF1, IF2, and IF3, the mRNA, and the fMet-tRNAfMet are shown in red, blue, green, yellow, and magenta, respectively. The components are placed on the ribosome according to current experimental knowledge. Details of the pathway are given in the text. Structures are derived from PDB entries as follows: 30S ribosomal subunit, 1HR0; 50S ribosomal subunit, 1FFK; IF1, 1HR0; IF2, 1G7T; IF3N, 1TIF; IF3C, 1TIG; mRNA, 1JGQ; fMet-tRNAfMet, 1JGQ. 

The bacterial 70S ribosome is composed of a large 50S and a small 30S subunit. It has three tRNA binding sites designated the aminoacyl (A), peptidyl (P), and exit (E) sites. Binding of IF3 to the 30S ribosomal subunit promotes dissociation of the ribosome into subunits and thus couples ribosome recycling and translation initiation. Initiation factor IF1 binds specifically to the base of the A-site of the 30S ribosomal subunit and is thought to direct the initiator tRNA to the ribosomal P-site by blocking the A-site. IF1 stimulates the activities of IF3 and hence also the dissociation of the ribosomal subunits.

Following subunit dissociation, IF2, mRNA, and fMet-tRNAfMet associate with the 30S ribosomal subunit in an unknown and possibly random order. The Shine-Dalgamo (SD) sequence of canonical mRNAs interacts with the anti-SD sequence of the 16S rRNA, and the initiation codon is adjusted in the P-site of the ribosome. The initiation factors (especially IF3) seem to be responsible for this adjustment. The initiator tRNA is positioned in the P-site of the 30S ribosomal subunit in three steps that are designated codon-independent binding, codon-dependent binding, and fMet-tRNAfMet adjustment. All three steps are probably promoted by IF2, which interacts with fMet-tRNAfMet on the ribosome. Furthermore, IF3 stabilizes the binding of fMet-tRNAfMet to the ribosomal P-site and confers proofreading capability by destabilization of a mismatched codon-anticodon interaction.

The 30S preinitiation complex consists of the 30S ribosomal subunit, the three initiation factors, and mRNA in a standby position where fMet-tRNAfMet is bound in a codon-independent manner. This relatively unstable complex undergoes a rate-limiting conformational change that promotes the codon-anticodon interaction and forms the more stable 30S initiation complex. Initiation factors IF1 and IF3 are ejected, while IF2 stimulates association of the 50S ribosomal subunit to the complex. Initiator fMet-tRNAfMet is adjusted to the correct position in the P-site, and IF2 is released from the complex. During this process, GTP bound to IF2 is hydrolyzed to GDP and Pi. The newly formed 70S initiation complex holding fMet-tRNAfMet as a substrate for the peptidyltransferase center of the 50S ribosomal subunit is ready to enter the elongation phase of translation.

COMPONENTS INVOLVED IN TRANSLATION INITIATION
The translation initiation event is a complex and highly regulated process involving both RNA and protein components. 

Stabilization of the ribosomal structure.


Two-thirds of the ribosome is composed of RNA. Three main types of interactions stabilize the tertiary structure of the rRNA: 

(i) Mg2+ bridges, 
(ii) RNA-RNA interactions, and 
(iii) RNA-protein interactions. 

The magnesium ions form neutralizing bridges between two or more phosphate groups from secondary-structure elements remote in sequence. RNA-RNA interactions of different types exist: (i) base pairing between nucleotides associated with secondary-structure elements remote in sequence, and (ii) A-minor motifs. The A-minor motif is an interaction between an adenine that inserts its minor groove face into the minor groove of a base pair in a helix. This is most often a GC pair. The adenine forms hydrogen bonds with one or both of the backbone 2′ hydroxyl groups of the RNA duplex. Different types of helix-helix packing interactions occur, involving the insertion of a ridge of phosphates into the minor groove of another helix or using an unpaired purine base to mediate the perpendicular packing of one helix against the minor groove of another.

RNA-protein interactions occur mainly via the sugar-phosphate backbone of the RNA. Thus, the ribosomal proteins recognize the unique shape of the rRNA rather than the bases, and the interactions are therefore sequence unspecific. Many of the ribosomal proteins have nonglobular extensions that are highly conserved in sequence. These tails penetrate into the ribosome and fill the gaps between RNA helices. In isolation, these protein tails, which contain approximately 26% arginine and lysine residues, look like random coils that probably only assume the conformation they have on the ribosome when bound (4, 219).

Prior to peptide bond formation, an aminoacyl-tRNA is bound in the ribosomal A-site, a peptidyl-tRNA is bound in the P-site, and a deacylated tRNA, which is ready for ejection from the ribosome, is bound to the E-site. Translation moves the tRNA from the A-site through the P- and E-sites before they exit the ribosome again, with the exception of the initiator tRNA, which binds directly to the P-site. The small ribosomal subunit contains the decoding center, where the triplet codons of the mRNA are base-paired with the anticodons of the cognate tRNA, and hence determines the sequence of amino acids to be incorporated in the synthesized protein. The large subunit contains the peptidyltransferase center and is thus the catalytic unit.

Small ribosomal subunit
The small ribosomal subunit is composed of 21 proteins and an RNA of approximately 1,500 nucleotides sedimenting at 16S. The shape of the subunit is determined largely by the RNA component, which forms four secondary-structure domains (Fig. ​(Fig.2)2).

Translation through ribosomes,  amazing nano machines - Page 2 Struct13
Structures of the ribosomal subunits.
(A) Overview of the 16S rRNA secondary structure. The domains are shown in colors according to the secondary structure: blue, 5′ domain (bulk of body); magenta, central domain (platform); red, 3′ major domain (head); yellow, 3′ minor domain (helices 44 and 45 located at the subunit interface).
(B) Overview of the 23S and 5S rRNA secondary structures. The RNA domains are shown in colors according to the secondary structure of the 23S rRNA: blue, domain I; cyan, domain II; green, domain III; yellow, domain IV; red, domain V; magenta, domain VI. The 5S rRNA is shown in orange.
(C) Three-dimensional structure of the 30S ribosomal subunit from T. thermophilus at 3-Å resolution (PDB entry 1J5E). RNA secondary-structure domains are colored as in panel A. Note that the secondary-structure domains of the RNA correspond well to the tertiary domains. Proteins are omitted for clarity. The tRNA binding sites A, P, and E are indicated.
(D) Three-dimensional structure of the 50S ribosomal subunit from H. marismortui at 2.4-Å resolution (PDB entry 1FFK). Colors are the same as in panel B. Note that the secondary-structure domains of the RNA do not correspond to the tertiary domains, unlike for the 30S subunit. Proteins are omitted for clarity. The L1 stalk, the central protuberance (CP), and the L7-L12 stalk are indicated.

Traditionally, the subunit has been divided into an upper third, called the head, connected by the neck to the body with a shoulder and platform. A protrusion in the lower part of the body is called the spur (or toe). The side of the 30S subunit facing the 50S subunit is called the front, whereas the solvent-exposed side is called the back. The small subunit binds mRNA and the anticodon loop and stem of tRNAs. Translational fidelity is controlled on the subunit by monitoring the base pairing between the codon and anticodon in the process known as decoding. The decoding center located at the upper part of the body and lower part of the head of the subunit is constructed entirely of RNA and contains, among other elements, the upper part of helix 44 and the 3′ and 5′ ends of the 16S rRNA. An interaction that is important for translation initiation occurs at the 3′ end of the 16S rRNA (also known as the anti-SD [ASD] sequence) that base-pairs with the SD sequence of the mRNA.

E. coli has 41 different tRNA species with different anticodons. The ribosome must select the tRNA with an anticodon complementary to the codon of the mRNA. This is termed the cognate tRNA. The error rate of tRNA selection in the decoding process is 1000 to 10.000. In addition to having lower dissociation rates from the ribosome, cognate tRNAs have higher rates of elongation factor EF1A GTPase activation and accommodation (movement of the aminoacyl end of tRNA into the A-site of the 50S ribosomal subunit) than do near-cognate tRNAs. Based on this result, it was proposed that binding of cognate tRNA induces a conformational change of the ribosome. Crystal structures of the 30S subunit complexed with mRNA and cognate tRNA in the A-site reveal an induced fit mechanism. Bases A1492 and A1493 of the 16S rRNA flip out of helix 44 and interact with the correctly base-paired codon-anticodon helix in an A-minor motif type interaction. A1492 and A1493 interact with the minor groove of a correctly paired codon-anticodon but not with incorrectly paired codons and anticodons. Binding of cognate tRNA also causes a flip of G530 of the 16S rRNA from syn to anti conformation. Bases A1492 and A1493 interact with the first and second base pairs of the codon-anticodon helix, respectively. G530 interacts with the second position of the anticodon as well as the third position of the codon. The result is a strictly monitored codon-anticodon interaction in the first two positions, whereas the ribosome is able to tolerate noncanonical base pairs at the third position. During decoding, the flipping of the 30S subunit bases A1492, A1493, and G530 translates to other parts of the subunit and leaves it in a closed conformation in which the shoulder and head domains are rotated toward the subunit center, compared to the more open structure when the A-site is unoccupied. The transition to the more closed state is unfavorable for near-cognate tRNAs. However, X-ray crystal structures of the intact 70S E. coli ribosome reveal that the closing of the head domain of the 30S subunit is connected to formation of the intact 70S ribosome and not to decoding. These structures do, however, indicate a movement of the small-subunit body connected with decoding. Thus, ribosomes play an active role in tRNA selection by direct recognition of the codon-anticodon base pairing.

Large ribosomal subunit.
The large ribosomal subunit is composed of 34 proteins and two RNAs sedimenting at 5S and 23S, containing about 120 and 2,900 nucleotides, respectively. Six secondary-structure domains are defined by the 23S rRNA, whereas the 5S rRNA is regarded as the seventh domain of the subunit. A direct relationship between secondary structure elements and morphological domains is not present in the large subunit, which presents a more compact structure than the small subunit (Fig. ​(Fig.2).2). The 50S subunit consists of a rounded base with three protuberances called the L1 protuberance, the central protuberance, and the L7/L12 stalk (Fig. ​(Fig.2)2). A tunnel starts at the peptidyltransferase center (PTC), where the formation of peptide bonds occurs. The nascent polypeptide is thought to exit at the base of the cytoplasmic side of the subunit through the approximately 100-Å-long tunnel, which has an average diameter of 15 Å.

During the peptidyl transfer reaction, the α-amino group of A-site tRNA attacks the carbonyl group of the P-site peptidyl group, which is linked to the tRNA via an ester bond. The reaction proceeds via a tetrahedral intermediate to form a peptidyl bond. The reaction occurs in the PTC, where the amino acid of the A-site tRNA has been properly positioned relative to the nascent peptide chain bound to the P-site tRNA. Peptide bond formation is then catalyzed. The PTC was identified by soaking crystals of Haloarcula marismortui 50S subunits with a transition state analogue, the so-called Yarus inhibitor. Surprisingly, the subunit is completely devoid of protein within 18 Å of the PTC, and the ribosome is thus a ribozyme. It is beyond the scope of this review to go into detail about the mechanism of the peptidyl transferase reaction. 

After the peptidyltransferase reaction has occurred, a deacylated tRNA is left in the P-site and the A-site tRNA is covalently bound to a peptide chain extended by one residue. For elongation to proceed, the P-site tRNA has to move into the E-site ready for ejection from the ribosome and the A-site peptidyl-tRNA has to move to the P-site. The E-site is specific for deacylated tRNAs. The movement of tRNAs must be accompanied by a precise movement of the mRNA to preserve the reading frame.

mRNA
mRNA interacts specifically with tRNA as well as the 30S ribosomal subunit during translation initiation. The mRNA covered by the ribosome in the translation initiation phase is called the ribosomal binding site (RBS) and extends over about 30 nucleotides. Bacterial mRNAs are normally polycistronic and possess multiple signals for initiation and termination of protein synthesis.

TIRs on mRNAs are not only characterized by the presence of a putative initiation codon. Additional elements are necessary to promote correct initiation and avoid initiation from, for instance, AUG codons encoding internal methionines of a protein. Upstream from the initiation codon is the 5′ untranslated region (5′ UTR). This region contains the SD sequence, which can undergo base-pairing to the 3′ of the 16S rRNA of the 30S ribosomal subunit. A direct consequence of the SD interaction is the adjustment of the initiation codon to the ribosomal P-site, where it interacts with fMet-tRNAfMet. E. coli mRNAs typically have the SD sequence GGAGG located 7 ± 2 nucleotides upstream from the initiation codon, which can be AUG, GUG or UUG. The exceptional AUU initiation codon has been observed in infC (encoding IF3) and pcnB [encoding E. coli poly(A) polymerase]. Initiation codons in E. coli occur at a frequency of 90, 8, and 1% for AUG, GUG, and UUG, respectively.

Ribosomal protein S1 interacts with a pyrimidine-rich region 5′ to the SD region on mRNAs. This pyrimidine-rich region acts as a ribosome recognition site. A direct interaction has been confirmed by cryoelectron microscopy (EM) studies of S1 on the 30S ribosomal subunit with a bound mRNA.

A region downstream from the initiation codon of several mRNAs was found to show complementarity to bases 1469 to 1483 within helix 44 of the 16S rRNA. This region was named the downstream box (DB), and there appeared to be a correlation between the degree of complementarity to the 16S rRNA and the translational efficiency of the mRNA. A mechanism similar to the SD-ASD interaction was proposed. The presence of a DB-anti-DB (ADB) interaction has been a matter of debate, and a recent review concludes that there is no biochemical or genetic evidence in support of the proposed role of the DB-ADB interaction in ribosomal recruitment of mRNA. This is supported by the crystal structure of the T. thermophilus 30S ribosomal subunit, which reveals that the shoulder of the subunit is located between the putative DB of the mRNA and the proposed anti-DB of the 16S rRNA .

Bacterial mRNAs are either canonical or leaderless, although the latter is rare, with no more than ∼40 identified cases in bacteria. Canonical mRNAs contain the 5′ UTR elements described above, whereas leaderless mRNAs start at, or a few nucleotides 5′ upstream of, the initiation codon. A clear mechanism for binding of canonical mRNAs and the order in which mRNA and fMet-tRNAfMet enter the 30S ribosomal subunit has not been established (Fig. ​(Fig.3).3).

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Binding of mRNA to the 30S ribosomal subunit.
(A) Binding of a canonical mRNA to the 30S ribosomal subunit. Two alternative pathways are shown where either the mRNA or the fMet-tRNAfMet binds first, followed by the other component. The mRNA is bound via the SD-ASD interaction as well as the codon-anticodon interaction.
(B) Binding of a leaderless mRNA to the 30S ribosomal subunit. The mRNA is bound to the ribosome mainly via the codon-anticodon interaction. IF2 stimulates the binding of leaderless mRNAs, presumably by recruitment of fMet-tRNAfMet to the subunit.

Initiation factors do not affect the SD-ASD interaction or the association between the 30S ribosomal subunit and canonical mRNA (61). However, site-directed cross-linking experiments have shown that mRNA is partially relocated on the 30S ribosomal subunit from a “standby site” to a site closer to the P-site in a process influenced by IF1, IF2, and especially IF3 .

Binding of leaderless mRNAs to the ribosome involves a mechanism that is somewhat different from binding of canonical mRNAs. The binding is dependent on the presence of the initiator tRNA, whereas canonical mRNAs bind independently of the initiator tRNA. Studies with E. coli revealed that the ratio of IF2 to IF3 plays an important role in translation initiation of leaderless mRNAs. Leaderless mRNA is recognized by a 30S-IF2-fMet-tRNAfMet complex equivalent to that formed during translation initiation in eukaryotes (Fig. ​(Fig.3)3). An increase in the concentration of IF2 enhances the efficiency of leaderless mRNA translation, possibly by recruitment of fMet-tRNAfMet to 30S ribosomal subunits, thus enabling codon-anticodon interaction. Recently, a cell-free translation system was used to show that leaderless mRNAs preferentially interact with 70S ribosomes and are able to proceed from the initiation to the elongation phase even in the absence of initiation factors.

The mRNA wraps around the neck of the 30S ribosomal subunit, with its 5′ end on the platform side and its 3′ end near the shoulder. Structural data for the interaction between mRNA and the ribosome are now available from X-ray crystallographic studies. The new data confirm the general features of the previous models. Interaction between the ASD and SD sequences is located at a large cleft between the head and the back of the platform on the 30S ribosomal subunit (Fig. ​(Fig.4).4).

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mRNA bound to the 30S ribosomal subunit.
(A) A 36-nucleotide mRNA is bound to the 30S ribosomal subunit. rRNA is shown in grey, mRNA is shown in yellow, and protein is shown in cyan. The ASD sequence of the 16S rRNA is shown in red to indicate the SD-ASD interaction. The P-site initiation codon is shown in green, and the A-site codon is shown in magenta. Note the kink in the mRNA between the two codons.
(B) Close-up of the region indicated in panel A. The upstream and downstream tunnels are marked by arrows. Colors are the same as in panel A. The structure is derived from PDB entry 1JGQ, prepared using the program Ribbons, and rendered in Pov-Ray.

The mRNA wraps around the 30S ribosomal subunit while it passes through the up- and downstream tunnels. A latch-like closure between the head and body on activation of the subunit forms the tunnels. Early studies indicated that binding of mRNA to the ribosome through the SD interaction melts the mRNA secondary structure in the TIR of the mRNA. The mRNA is probably unwound by mRNA helicases before entering the downstream tunnel, since an RNA helix is too large to pass.

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

Translation through ribosomes,  amazing nano machines - Page 2 Initia10
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.

Selection of the correct AUG in a prokaryotic mRNA requires sequences in the mRNA called Shine-Dalgarno sequences (FIGURE). The ribosome is properly positioned at the correct AUG by base pairing between sequences in the 16S rRNA and the Shine-Dalgarno sequences in the mRNA. Messages with mutated Shine-Dalgarno sequences are poorly translated.

The correct AUG in a eukaryotic mRNA is almost always the first AUG from the 5'-end, but the context of the surrounding sequence is important: GCC(A or G)CCAUGG. The most important features of this sequence are the purine (A or G) 3 bases before the AUG and the G immediately following it (together they influence the efficiency of translation by 10-fold).

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

Translation through ribosomes,  amazing nano machines - Page 2 The_sh10
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.

The Initiation Complex Assembles
Before protein synthesis starts, the two subunits of the ribosome are floating around separately. Because the 16S rRNA, with the anti-Shine-Dalgarno sequence, is in the small subunit of the ribosome, the mRNA binds to a free small subunit. Next the initiator tRNA, carrying fMet, recognizes the AUG start codon. Assembly of this 30S initiation complex needs three proteins (IF1, IF2, and IF3), known as initiation factors, which help arrange all the components correctly. IF2 physically contacts the acceptor stem of the fMet-tRNA, and this interaction is essential to stabilize the initiation complex. IF3 recognizes the start codon and the matching anticodon end of the initiator tRNA. IF3 prevents the 50S subunit from binding prematurely to the small subunit before the correct initiator tRNA is present. Once the 30S initiation complex has been assembled, IF3 departs and the 50S subunit binds. IF1 and IF2 are now released, resulting in the 70S initiation complex.

Proteins known as initiation factors help the ribosomal subunits, mRNA and tRNA assemble correctly.

Translation through ribosomes,  amazing nano machines - Page 2 Format10
Formation of 30S and 70S Initiation Complexes
(A) The small subunit and the mRNA bind to each other at the Shine-Dalgarno sequence. The start codon, AUG, is just downstream of this site.
(B) The initiator tRNA becomes tagged with fMet and binds to the AUG codon on the mRNA.
(C) The large ribosomal subunit joins the small subunit and accommodates the tRNA at the P-site.

This process consumes energy in the form of GTP, which is split by IF2.

Initiation of Protein Synthesis in eukaryotes


Regulation of cap-dependent translation by eIF4E inhibitory proteins 3 FEBRUARY 2005   1
Eukaryotic messenger RNAs contain a modified guanosine, termed a cap, at their 5' ends. Translation of mRNAs requires the binding of an initiation factor, eIF4E, to the cap structure. Here, we describe a family of proteins that through a shared sequence regulate cap-dependent translation. The biological importance of this translational regulation is immense, and affects such processes as cell growth, development, oncogenic transformation and perhaps even axon pathfinding and memory consolidation.

A single cell never exploits the full panoply of gene products available for its use; at any one time, the transcription of most genes is unwarranted and therefore these genes are silenced. Even for those mRNAs that are synthesized and transported to the cytoplasm, however, there are often other levels of regulation. The past few years have witnessed an explosion in the number of mRNAs whose translation is recognized to be temporally and spatially regulated in various cell types. Although no single mechanism controls the translation of all mRNAs, emerging evidence indicates that the regulated binding of translation initiation factors (eIFs) to the 7-methyl guanosine residue that caps the 50 ends of all nuclear-encoded eukaryotic mRNAs is important. In particular, the interaction of the ribosomal-subunit-associated eIF4G with the cap-bound eIF4E is necessary for cap-dependent translation. A group of factors generically known as eIF4E inhibitory proteins modulate the eIF4G–eIF4E interaction. Whereas some eIF4E inhibitory proteins repress translation by associating with eIF4E on a large number of transcripts, others are tethered ( restrict its movement) to specific subsets of mRNAs through interactions with RNA binding proteins, thus restricting their inhibition of translation to only certain mRNAs. Biologically, the eIF4E inhibitory proteins are enormously important; they control development and cell growth, repress tumour formation, and may influence critical neuronal events such as axon guidance and synaptic plasticity, a phenomenon that may underlie long-term memory storage. Here, we present not only the molecular mechanisms by which some of these proteins control translation, but also describe a few of the biological processes they regulate. Although only a handful of these eIF4E inhibitory proteins have been identified, we suspect that there may be several others that await discovery

The translation initiation machinery 
Initiation is the rate-limiting step in translation and is the most common target of translational control. The mRNA 50 cap is bound by eIF4F, a heterotrimeric protein complex that is the focal point for initiation. eIF4G is the backbone of this complex; it interacts not only with eIF4E, but also with eIF4A, an RNA helicase that facilitates ribosome binding and its passage along the 50 untranslated region (UTR) towards the initiation codon. eIF4G also associates with eIF3, a multisubunit factor that bridges the proteins bound to the mRNA’s 50 end with the 40S ribosomal subunit (Fig. 1). 

Translation through ribosomes,  amazing nano machines - Page 2 1_tran12

This ribosomal subunit comes ‘pre-charged’ as a ternary complex composed of eIF2, GTP and the initiator methionine-transfer RNA. With the aid of eIF4 initiation factor as well as ATP, this agglomeration of RNA and protein is thought to scan the mRNA in the 50 to 30 direction. When it encounters an AUG start codon in an optimal context, other factors as well as the 60S ribosomal subunit are recruited and polypeptide chain elongation begins . The eIF4E–eIF4G interface is an important target for translational control. The core portion of eIF4G that interacts with eIF4E is small—about 15 amino-acid residues . Strikingly, several other proteins contain similar peptide motifs, and it is this region that competes with eIF4G for binding to eIF4E; in this manner they control the rate of 40S ribosomal subunit association with mRNA, and hence translation initiation. Peptides derived from the regions of eIF4G and an eIF4E inhibitory protein called 4E-BP (for 4E-binding proteins, also known as PHAS-I for phosphorylated heat and acid soluble protein stimulated by insulin) form nearly identical a-helical structures that lie along the same convex region of eIF4E, some distance from this protein’s cap binding site. Peptides with the general sequence YXXXXLf, where f is any hydrophobic amino acid, would probably form similar a-helical structures, implying that other proteins containing this peptide motif could control translation initiation. The original three eIF4E inhibitory proteins, the 4E-BPs, prevent eIF4F complex formation by sequestering available eIF4E. This sequestration results in the inhibition of translation of certain mRNAs that normally require high levels of available eIF4E. The newly discovered eIF4E-binding proteins described below interact with the eIF4E on only specific mRNAs, and do so either because they also interact with certain RNA elements directly, or do so through affiliations with RNA binding proteins.







1.https://www.researchgate.net/profile/Nahum-Sonenberg/publication/8042588_Regulation_of_cap-dependent_translation_by_eIF4E_inhibitory_proteins/links/0912f51467599db728000000/Regulation-of-cap-dependent-translation-by-eIF4E-inhibitory-proteins.pdf



Last edited by Otangelo on Tue Mar 16, 2021 7:36 am; edited 5 times in total

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27Translation through ribosomes,  amazing nano machines - Page 2 Empty Elongation Factor Tu Thu Feb 11, 2021 10:37 am

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Elongation Factor Tu

https://reasonandscience.catsboard.com/t1661p25-translation-through-ribosomes-amazing-nano-machines#8442

https://www.youtube.com/watch?v=2guXr5c4rHc
We've heard about called ef-tu ef-tu binds to the aminoacyl part of the trna and protects it from actually interacting with the decoding I mean with the peptidyl transferase region ef-tu also has other functions of forsee it facilitates binding the synthetase and putting the amino acid on here and ef tu also has an affinity for the 50s subunit at 60s subunit so it's actually bringing the tRNA to the ribosome but it's protecting it at the same time protecting this until you're certain that you have the right tRNA

In the elongation cycle of protein biosynthesis in bacteria and eukaryotic organelles, one of the most essential steps is the formation of an active ternary complex between elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA) and GTP, after which the aa-tRNA is delivered to the ribosomal A-site 1

EF-Tu and EF-Ts are required for binding aminoacyl-tRNAs to the ribosome 2

EF-Tu (elongation factor thermo unstable) is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome.
https://en.wikipedia.org/wiki/EF-Tu#:~:text=EF-Tu%20(elongation%20factor%20thermo,A-site%20of%20the%20ribosome.

Recognition and selection of tRNA in translation 7 February 2005
https://www.sciencedirect.com/science/article/pii/S0014579304014310
The ribosome is a large ribonucleoprotein complex that catalyzes the translation of a messenger RNA (mRNA) into protein. During elongation of the growing polypeptide, the mRNA codons are displayed one after another and aminoacyl-tRNAs (aa-tRNA) with complementary anticodons are selected. Elongation factor Tu (EF-Tu) delivers aa-tRNA to the ribosomal aa-tRNA binding site (A site) where tRNA recognition and selection takes place. Biochemical and kinetic studies have shown that the movement of aa-tRNA into the A site proceeds through a number of intermediate states

Translation through ribosomes,  amazing nano machines - Page 2 1-s2_012

Kinetic mechanism of EF-Tu-dependent aa-tRNA binding to the A site. 
Kinetically resolved steps are indicated by rate constants k1 − k7, k−1, k−2 and the two chemical steps that are rate-limited by the preceding step are designated kGTP and kpep. . EF-Tu is depicted in different conformations in the GTP- and GDP-bound form and in the activated GTPase state. Dissociation of EF-Tu (k6) (see text) is not shown for simplicity; it takes place concomitantly with, and independently of, aa-tRNA accommodation and rejection. The codon recognition step can be subdivided into (i) the codon reading by aa-tRNA (0.35 FRET;) and (ii) the formation of the interactions of the ribosome with the codon–anticodon duplex (0.5 FRET;). The GTPase activation step can be distinguished from the codon recognition step by biochemical and stopped-flow kinetic experiments, but does not result in a smFRET change (0.5 FRET;).


A GTP-Binding Protein Shows How Large Protein Movements Can Be Generated
Detailed structures obtained for one of the GTP-binding protein family members, the EF-Tu protein, provide a good example of how allosteric changes in protein conformations can produce large movements by amplifying a small, local conformational change. EF-Tu is an abundant molecule that serves as an elongation factor (hence the EF) in protein synthesis, loading each aminoacyl-tRNA molecule onto the ribosome. EF-Tu contains a Ras-like domain, and the tRNA molecule forms a tight complex with its GTP-bound form. 

Translation through ribosomes,  amazing nano machines - Page 2 Ras10
The structure of the Ras protein in its GTP-bound form. 
This monomeric GTPase illustrates the structure of a GTP-binding domain, which is present in a large family of GTP-binding proteins. The red regions change their conformation when the GTP molecule is hydrolyzed to GDP and inorganic phosphate by the protein; the GDP remains bound to the protein, while the inorganic phosphate is released.

This tRNA molecule can transfer its amino acid to the growing polypeptide chain only after the GTP bound to EF-Tu is hydrolyzed, dissociating the EF-Tu. Since this GTP hydrolysis is triggered by a proper fit of the tRNA to the
mRNA molecule on the ribosome, the EF-Tu serves as a factor that discriminates between correct and incorrect mRNA–tRNA pairings

Translation through ribosomes,  amazing nano machines - Page 2 Detail11
Translation through ribosomes,  amazing nano machines - Page 2 Detail10
Detailed view of the translation cycle. 
The outline of translation has been expanded to show 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

By comparing the three-dimensional structure of EF-Tu in its GTP-bound and GDP-bound forms, we can see how the repositioning of the tRNA occurs.

Bacterial elongation factor EF-Tu
https://www.youtube.com/watch?v=6vKk7LEf6Rk

The dissociation of the inorganic phosphate group (Pi), which follows the reaction GTP → GDP + Pi, causes a shift of a few tenths of a nanometer at the GTP-binding site, just as it does in the Ras protein. This tiny movement, equivalent to a few times the diameter of a hydrogen atom, causes a conformational change to propagate along a crucial piece of α helix, called the switch helix, in the Ras-like domain of the protein. The switch helix seems to serve as a latch that adheres to a specific site in another domain of the molecule, holding the protein in a “shut” conformation. The conformational change triggered by GTP hydrolysis causes the switch helix to detach, allowing separate domains of the protein to swing apart, through a distance of about 4 nm. 

Translation through ribosomes,  amazing nano machines - Page 2 The_la10
The large conformational change in EF-Tu caused by GTP hydrolysis. 
(A and B) The three-dimensional structure of EF-Tu with GTP bound.The domain at the top has a structure similar to the Ras protein, and its red α helix is the
switch helix, which moves after GTP hydrolysis. (C) The change in the conformation of the switch helix in domain 1 allows domains 2 and 3 to rotate as a single unit by about 90 degrees toward the viewer, which releases the tRNA that was bound to this structure

Translation through ribosomes,  amazing nano machines - Page 2 An_ami10
An aminoacyl tRNA molecule bound to EF-Tu.
Note how the bound protein blocks the use of the tRNA-linked amino acid (green) for protein synthesis until GTP hydrolysis triggers the conformational changes shown in Figure 3–72C, dissociating the protein-tRNA complex.
EF-Tu is a bacterial protein; however, a very similar protein exists in eukaryotes, where it is called EF-1



This releases the bound tRNA molecule, allowing its attached amino acid to be used (Figure 3–73).







EF-Tu participates in the polypeptide elongation process of protein synthesis. In prokaryotes, the primary function of EF-Tu is to transport the correct aa-tRNA to the A-site of the ribosome. As a G-protein, it uses GTP to facilitate its function. Outside of the ribosome, EF-Tu complexed with GTP (EF-Tu • GTP) complexes with aa-tRNA to form a stable EF-Tu • GTP • aa-tRNA ternary complex.

Chaperone Properties of Bacterial Elongation Factor EF-Tu January 30, 1998)
https://www.jbc.org/article/S0021-9258(19)89346-8/pdf
There is a high level of EF-Tu in Escherichia coli cells, comprising 5–10% total cell protein, in vast molar excess over the other essential protein components of the translation machinery. Several sets of results suggest that EF-Tu and its eukaryotic counterpart EF1a may have other functions in addition to the conventional role that they play in polypeptide elongation. EF-Tu is an essential host-donated subunit of the replicative complex of Qb phage (4), and it may interact with the transcriptional apparatus as a positive regulator of RNA synthesis (5). EF-Tu is associated in part with the plasma membrane (6)




Translation through ribosomes,  amazing nano machines - Page 2 Ch7f11
Elongation stage of translation
The ribosome has three tRNA-binding sites, designated P (peptidyl), A (aminoacyl), and E (exit). The initiating N-formylmethionyl tRNA is positioned in the P site, leaving an empty A site. The second aminoacyl tRNA (e.g., alanyl tRNA) is then brought to the A site by EF-Tu (complexed with GTP). Following GTP hydrolysis, EF-Tu (complexed with GDP) leaves the ribosome, with alanyl tRNA inserted into the A site. A peptide bond is then formed, resulting in the transfer of methionine to the aminoacyl tRNA at the A site. The ribosome then moves three nucleotides along the mRNA. This movement translocates the peptidyl (Met-Ala) tRNA to the P site and the uncharged tRNA to the E site, leaving an empty A site ready for addition of the next amino acid. Translocation is mediated by EF-G, coupled to GTP hydrolysis. The process, illustrated here for prokaryotic cells, is very similar in eukaryotes.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1188084/
2. https://uh.edu/~phardin/MolBioCh18-04.html



Last edited by Otangelo on Thu Mar 18, 2021 3:03 pm; edited 15 times in total

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200 other non-ribosomal proteins and 75 snoRNAs are required for ribosome biogenesis.
Ribosome synthesis and assembly is highly regulated, 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 

The RA-GTPases are universally conserved proteins that couple GTP hydrolysis to specific checkpoints in assembly.
Ribosomes are complexes of precisely folded RNA and proteins.
The ways in which the ribosome production and assembly are managed by the living cell is of deep biological importance.
It can be tempting  to think of the bacterial cell as a finely tuned machine for building ribosomes.
Ribosome production is tightly regulated by the cell.  
coordinated transcription, translation, folding, and hierarchical assembly
hierarchical association of sets of ribosomal proteins
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 
Ribosome 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.  
fine-tune rRNA structure or function
The role of these factors is to drive ribosome assembly in a unidirectional manner. 
The assembly of a number of ribosomal proteins into ribosome intermediates is aided by dedicated chaperones
escort them into the nucleus or help insert them into pre-ribosomal particles undergoing assembly. 
allowing each domain to be assembled separately in an encapsulated environment. 
which occurs in a modular fashion
snoRNA provides a crucial spatial constraint that dictates the topology of the maturing particle.  
The yeast SSU processome provides an encapsulated environment for the rRNA subdomains of the small subunit.
Ribosome assembly factors predominantly act as local stabilizers of RNA elements. 
numerous protein exchanges enable other major remodelling events, leading to export from the nucleus. 
the removal of remaining assembly factors, assembly of the last few ribosomal proteins and test-driving of functional centres.
Thus, proper folding of ITS1 sequences is necessary to signal successful initiation of assembly of the large subunit .  
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.
The 14-subunit exosome removes.......
then rotates ~180° to assume its mature configuration.
central protuberance helices undergo a large- scale structural rearrangement. 
yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly 
Arx1 binding may serve to proofread the proper accommodation of these ribosomal proteins into the tunnel exit
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. 
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.  
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. 
The C-terminal domain of uS3  orchestrates simultaneous nuclear import and protection of this r protein
the trimeric Syo1–uL18–uL5 cargo complex is released in a RanGTP-dependent manner. 
eS26, like a typical import cargo, directly binds to importins for targeting to the nucleus. 
The second mechanism involves the prefabrication of ribosomal protein complexes
The degradation pathway, termed ERISQ, for excess ribosomal protein quality control, may be one mechanism employed to maintain cellular homeostasis of r proteins.
The 90S assembly process is aided by ~70 assembly factors and small nucleolar RNAs (snoRNAs)
proteins that join the nascent 5′ ETS  in a sequential manner.
UtpA is composed of seven subunits,
This process is regulated by the TOR1 pathway
Prior to nuclear export, pre-40S subunits undergo an essential maturation step.
A myriad of assembly factors are implicated in nuclear pre-60S maturation. 
the remaining 5S RNP is recruited, and the pre-60S is compacted into its characteristic structure. 
The foot structure is established through an intricate interaction network 
This dramatic remodeling step
These two factors form a checkpoint for acquisition of export competence 
stably anchored through multiple contact points 
the exportin Crm1 playing an essential role the transport
To initiate export, Crm1 needs to recognize a nuclear export signal (NES) 
essential export adaptor protein 
NES that is essential for yeast viability
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
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
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.
Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. 
In contrast to prokaryotes, eukaryotic ribosome assembly requires coordinated efforts of the intracellular transport machinery as well as numerous transiently interacting nonribosomal assembly factors.
Assembly of the 90S appears to be a hierarchical addition of pre-formed protein sub-complexes
More than 60 different snoRNPs mediate >100 covalent modifications of the 35S pre-rRNA during the assembly of the 90S
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. 
a multitude of energy-consuming enzymes. 
confer directionality to the assembly and maturation process.
Three essential AAA-ATPases contribute to pre-60S subunit maturation. 
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. 
essential nuclear export signal (NES) containing adaptor 
Notably, the essential general mRNA transport receptor
Additionally, pre-ribosomal particles employ multiple nonessential, auxiliary factors 
These steps are not only crucial for completing ribosome maturation, but are also crucial for new rounds of ribosome biogenesis. 
crucial for assembly of the ribosome stalk,
efficient quality control system must ensure that only functional ribosomes enter translation
TRAMP complex marks and targets aberrant pre-rRNAs for degradation 
nuclear surveillance mechanisms are segregated and targeted for degradation 
Ribosomal subunits may sense their decoding ability to segregate and target aberrant particles for disassembly and degradation. 
represent a quality control mechanism that simultaneously triggers subunit maturation and senses translation competence.
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 
a surprisingly large number (>150) of accessory proteins and dozens of snoRNAs are involved.
the interdependency in rRNA processing acts, at least in part, as a mechanism which helps assure that only functional RNA is incorporated. 
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.
 a ribosome assembly chaperone (RAC), presumably acting as a kind of rack on which critical structure is organized. 
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. 
During pre-40S maturation, formation of essential contacts between Rps20 and Rps3
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.
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.
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.
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.
Several hundred maturation factors participate in ribosome biogenesis, and it has to be an immense logistic challenge to coordinate their action
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 Assembly Factors
Interestingly, additional examples lead to the proposal that communication across nascent ribosomal subunits may play a role in coordinating ribosome biogenesis
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.
c-MYC and the components of the PI3K-mTORC1 signaling pathway are emerging as key regulators of ribosome biogenesis.  
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. 
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 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).
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.

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’. 
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)
The oncoprotein c-Myc positively controls cell growth and proliferation and serves as a direct regulator of ribosome biogenesis
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
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.

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. 

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.

Eukaryotic ribosome biogenesis at a glance.
Ribosome biogenesis encompasses six important steps

(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. 

80 RPs, more than 250 Assembly Factors 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 undergo a ‘test drive’ to prove functionality before final maturation
Synthesis involves all three RNA polymerases (Pol I–III)
There are over 200 trans-acting AFs in budding yeast. 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
By design, their displacement is a prerequisite for catalytic activation of the ribosome. 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. T
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.


The conserved boxes are bound by proteins important for snoRNA stability, nucleolar targeting and snoRNP function. 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.

 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.
there is abundant cross-talk in the assembly of distinct RNPs, which sometimes rely on the same AFs. 

 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.

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Over 200 other non-ribosomal proteins and 75 snoRNAs are required for ribosome biogenesis.

Ribosome synthesis and assembly is highly regulated, 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 

The RA-GTPases are universally conserved proteins that couple GTP hydrolysis to specific checkpoints in assembly.
Ribosomes are complexes of precisely folded RNA and proteins
The ways in which the ribosome production and assembly are managed by the living cell is of deep biological importance.
It can be tempting  to think of the bacterial cell as a finely tuned machine for building ribosomes
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, and proceeds through the hierarchical association of sets of ribosomal proteins, each progressively folding and stabilizing the rRNA's growing tertiary structure. 
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!!
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  
Ribosome 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.  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
The role of these factors is to drive ribosome assembly in a unidirectional manner. 
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. 
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 large subunit maturation, which occurs in a modular fashion, where subdomains are bound by more-isolated assembly factors. 
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.  The yeast SSU processome provides an encapsulated environment for the rRNA subdomains of the small subunit.
Ribosome assembly factors predominantly act as local stabilizers of RNA elements. 
During the following transition from the nucleolus to the nucleoplasm, 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.
Thus, proper folding of ITS1 sequences is necessary to signal successful initiation of assembly of the large subunit .  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.
Nucleoplasmic stages of large-subunit assembly. 
The 14-subunit exosome removes.......
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 undergo a large- scale structural rearrangement. 
To minimize production of dysfunctional ribosomes, yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly 
before export of pre- ribosomal particles, Arx1 binding may serve to proofread the proper accommodation of these ribosomal proteins into the tunnel exit
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. 
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.  
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. 
The C-terminal domain of uS3  orchestrates simultaneous nuclear import and protection of this r protein
the trimeric Syo1–uL18–uL5 cargo complex is released in a RanGTP-dependent manner. 
eS26, like a typical import cargo, directly binds to importins for targeting to the nucleus. 
The second mechanism involves the prefabrication of ribosomal protein complexes
The degradation pathway, termed ERISQ, for excess ribosomal protein quality control, may be one mechanism employed to maintain cellular homeostasis of r proteins.
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.
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.
This process is regulated by the TOR1 pathway
Prior to nuclear export, pre-40S subunits undergo an essential maturation step.
myriad of assembly factors are implicated in nuclear pre-60S maturation. In addition, the remaining 5S RNP is recruited, and the pre-60S is compacted into its characteristic structure. 
The foot structure is established through an intricate interaction network involving the assembly factors Nop15, Cic1, Rlp7, Nop7 and Nop53. 
This dramatic remodeling step is likely carried out through recruitment of the Rix1 subcomplex. 
These two factors form a checkpoint for acquisition of export competence (
Only after accurate 5S RNP rotation can the huge Rix1–Rea1 machinery be stably anchored through multiple contact points on the pre-60S. 
In growing yeast cells, preribosomal particles are exported into the cytoplasm, with the exportin Crm1 playing an essential role the transport
To initiate export, Crm1 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 that is essential for yeast viability
Cytoplasmic maturation also provides a time window for the quality-control machinery to functionally proofread preribosomesIn the case of pre-60S subunits, energy-consuming enzymes trigger these steps in a stepwise fashion
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
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.
Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. 
In contrast to prokaryotes, eukaryotic ribosome assembly requires coordinated efforts of the intracellular transport machinery as well as numerous transiently interacting nonribosomal assembly factors. 
Assembly of the 90S appears to be a hierarchical addition of pre-formed protein sub-complexes
More than 60 different snoRNPs mediate >100 covalent modifications of the 35S pre-rRNA during the assembly of the 90S
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. 

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.
Three essential AAA-ATPases contribute to pre-60S subunit maturation. 
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. 
Subsequently, a visual screen and an independent genetic approach uncovered an essential nuclear export signal (NES) containing adaptor 
Notably, the essential general mRNA transport receptor Mex67-Mtr2 and the HEAT-repeat containing protein Rrp12 contribute to export of both pre-ribosomal subunits 
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
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.
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.
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)
Ribosomal subunits may sense their decoding ability to segregate and target aberrant particles for disassembly and degradation. 
Processing of the 20S pre-rRNA may represent a quality control mechanism that simultaneously triggers subunit maturation and senses translation competence.
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. 
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. 
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. 
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. 
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. 
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. 
During pre-40S maturation, formation of essential contacts between Rps20 and Rps3
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.
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.
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.
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.
Several hundred maturation factors participate in ribosome biogenesis, and it has to be an immense logistic challenge to coordinate their action
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 Assembly Factors
Interestingly, additional examples lead to the proposal that communication across nascent ribosomal subunits may play a role in coordinating ribosome biogenesis
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
c-MYC and the components of the PI3K-mTORC1 signaling pathway are emerging as key regulators of ribosome biogenesis.  
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. 
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 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)
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.

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’. 
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)
The oncoprotein c-Myc positively controls cell growth and proliferation and serves as a direct regulator of ribosome biogenesis
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
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.

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. 

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.

Eukaryotic ribosome biogenesis at a glance. 
Ribosome biogenesis encompasses six important steps : 

(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. 

80 RPs, more than 250 Assembly Factors 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 undergo a ‘test drive’ to prove functionality before final maturation
Synthesis involves all three RNA polymerases (Pol I–III)
There are over 200 trans-acting AFs in budding yeast. These and the many snoRNAs (75 in yeast, ~200 in humansrequired 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
By design, their displacement is a prerequisite for catalytic activation of the ribosome. 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. T
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.


The conserved boxes are bound by proteins important for snoRNA stabilitynucleolar targeting and snoRNP function. 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.

 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.
there is abundant cross-talk in the assembly of distinct RNPs, which sometimes rely on the same AFs. 

 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.











Understanding the biogenesis of the Ribosome

https://reasonandscience.catsboard.com/t1661p25-translation-through-ribosomes-amazing-nano-machines#8448

Understanding the biogenesis of the Ribosome is probably one of the most intellectually challenging tasks for anyone. If dedicated exclusively to this study, it will take several years to have a superficial and rudimentary understanding of the biochemical process. I have started to read some science papers, and the technical jargon is just over my mind. In order to know what is being said, one has to  understand the operation and function of each of the hundreds and hundreds of Assembly Factors, small nucleolar RNA's, ribosomal subunits, chaperones, and learn, how they are individually synthesized, and the entire assembly process. But some key words appear frequently. They give light about what goes on. Consider that all these processes must be pre-programmed to occur autonomously, in a robot-like fashion, without any external intervention whatsoever. 

anchoring,
biogenesis program,
cargo-shuttling, 
check-point control,
check-list ensurance,
communicating,
cross-talking
dedicating, 
efficiency,
exporting,
fabricating, 
fine-tuning, 
hierarchy, 
importing,
inspecting,
logistics, 
maturating,
network interaction, 
orchestrating,
organizing, 
precision,
processing, 
proof-reading,
quality control,
rearranging, 
recognizing, 
reorganizing, 
regulating, 
requirements, 
recruiting, 
remodeling, 
rotating, 
sequence,
sensing, 
signal-recognition, 
targeting, 
tight-coupling

Question: How could somebody in its sane mind conclude that these extremely complex and orderly assembly processes could be the product of stochastic chemical reactions on a prebiotic earth?  
 
Regulation of when and how much of the protein is produced  can be mind-bogglingly complex, involving dozens of other proteins and their interactions with DNA:

Regulation
Ribosome production is tightly regulated by the cell. 
This process is regulated by the TOR1 pathway
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.  (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 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.
Thus, ribosome biogenesis is highly regulated with diverse checkpoints to limit the production of altered ribosomes.
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
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.


Check-points
The RA-GTPases are universally conserved proteins that couple GTP hydrolysis to specific checkpoints in assembly.
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 Assembly Factors.
Thus, ribosome biogenesis is highly regulated with diverse checkpoints to limit the production of altered ribosomes.
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.

Proof-reading
Yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly
Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. 
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.
To minimize production of dysfunctional ribosomes, yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly
Arx1 binding may serve to proofread the proper accommodation of these ribosomal proteins into the tunnel exit
time window for the quality-control machinery to functionally proofread preribosomes. 


Quality-control
The degradation pathway, termed ERISQ, for excess ribosomal protein quality control, may be one mechanism employed to maintain cellular homeostasis of r proteins.
Time window for the quality-control machinery to functionally proofread preribosomes
Efficient quality control systems must ensure that only functional ribosomes enter translation
Represent a quality control mechanism that simultaneously triggers subunit maturation and senses translation competence.
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.

Precision
Ribosomes are complexes of precisely folded RNA and proteins.
critical task for the ribosome assembly machinery is to precisely incorporate r proteins onto dynamically folding pre-rRNA.

Efficiency
Ribosome assembly requires the efficient processing of the precursor rRNA transcripts followed by the ordered assembly of ribosomal proteins 
 the splicing apparatus and the cellular transport system are required to ensure highly efficient and accurate ribosome biogenesis.
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. 
Given the importance to correctly translate proteins, an efficient quality control system must ensure that only functional ribosomes enter translation. 
Ribosome biogenesis depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. [/size

Fine-tuning
It can be tempting  to think of the bacterial cell as a finely tuned machine for building ribosomes. 
[size=13]Ribosome biogenesis represents the most expensive, complexfinely tuned, multi-step process that the cell must carry-out


Hierarchy
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, and proceeds through the hierarchical association of sets of ribosomal proteins, each progressively folding and stabilizing the rRNA's growing tertiary structure.
Assembly of the 90S appears to be a hierarchical addition of pre-formed protein sub-complexes. 
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. 

Requirements
Ribosome assembly requires the efficient processing of the precursor rRNA transcripts followed by the ordered assembly of ribosomal proteins
The maturation of pre- rRNAs for both subunits requires endonucleolytic and exonucleolytic cleavage  
r-protein components, eukaryotic ribosomal subunit assembly requires >350 nonribosomal factors
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

In contrast to prokaryotes, eukaryotic ribosome assembly requires coordinated efforts of the intracellular transport machinery as well as numerous transiently interacting nonribosomal assembly factors. 
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.
Assembly of the snoRNPs themselves requires dozens of AFs. [/size]

Orchestrating
The C-terminal domain of uS3  orchestrates simultaneous nuclear import and protection of this r protein
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. 

Exporting
During the following transition from the nucleolus to the nucleoplasm, 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.
To minimize production of dysfunctional ribosomes, yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly  before export of pre- ribosomal particles, Arx1 binding may serve to proofread the proper accommodation of these ribosomal proteins into the tunnel exit
Prior to nuclear export, pre-40S subunits undergo an essential maturation step.
These two factors form a checkpoint for acquisition of export competence 
To initiate export, Crm1 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 that is essential for yeast viability. 

Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes.
Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. 



Last edited by Otangelo on Mon Mar 15, 2021 12:26 pm; edited 8 times in total

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Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster
https://www.mdpi.com/1422-0067/20/11/2718/htm
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.). This review examines some of the well-studied pathways known to control ribosome biogenesis (PI3K-AKT-mTOR, RB-p53, MYC) and how they may 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). 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.

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. This review aims to examine the major signal transduction events controlling ribosome biogenesis and the initiation of protein synthesis in higher eukaryotes. The role of well-studied pathways in ribosome biogenesis, such as the avian myelocytomatosis viral oncogene homolog (MYC)/MYC-associated factor X (MAX), mouse/human double minute 2 homolog (M/HDM2)-p53, and the phosphotidylinositol-3 kinase (PI3K)-AKR mouse thyoma homologue (AKT)-mammalian target of rapamycin (mTOR) pathways, will be reviewed as well as the roles of the less well studied eukaryotic initiation factor (eIF)-2α kinase (namely protein kinase R (PKR)) and RNA editing/alternate splicing, and how these pathways cross-talk to regulate ribosome biogenesis. Pathologies resulting from perturbations in these pathways will also be discussed.

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 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.

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 (Figure 1). 

Translation through ribosomes,  amazing nano machines - Page 2 Ijms-210
Figure 1. 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.

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 ribosomal proteins (RPs) are processed and transported to the cytoplasm for translation. RNA splicing of these transcripts also produces the Small Cajal body-specific RNAs (scaRNAs) and small nucleolar RNAs (snoRNAs) later needed for the formation of diverse heterogeneous ribonucleoprotein (hnRNP) complexes, C/D box snoRNAs (SNORDs) that are an abundantly expressed class of short, non-coding RNAs that have been long known to perform 2′-O-methylation of rRNAs , and H/ACA snoRNPs ( The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis 1

H/ACA snoRNPs function in mRNA splicing and rRNA modification/maturation (Figure 1). Alternative splicing of RP transcripts also produces “pseudogenes” that regulate the expression/accumulation of RPs (see section on RNA editing and splicing). 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. Recent developments in cryogenic-electron microscopy (cryo-EM) have shed a tremendous amount of light on the process of RP nuclear import and their incorporation into the ribosome.(Figure 1).

Names: 
ribosomal proteins (RPs)
ribonucleoprotein particle (RNP)
small subunit ribosomal proteins RPs or RPSs
large ribosomal subunit (RPLs)

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 ribosomal proteins (RPs) forming the 90S  ribonucleoprotein particle (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 ribosomal protein RPS7 and ribosomal protein RPS24, which are required to initiate processing and cleavage of the pre-rRNA at the 5′-external transcribed spacer (ETS) (Figure 1).


Translation through ribosomes,  amazing nano machines - Page 2 The_ri11


The initial binding of the ribosomal proteins (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  large ribosomal subunit (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 (Figure 1).

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.

Technological advances in microscopy have made it clear that the cell goes to great lengths in order to compartmentalize the phases of ribosome biogenesis. The nucleolus contains several sub-compartments

The fibrillar centers (FCs), 
the dense fibrillar centers (DFCs), and 
the granular compartments (GCs). 
In addition, on the nucleolus/nucleoplasm border there are the Cajal bodies, a distinct subcellular component responsible for non-coding RNA maturation

The rDNA clusters are located at the FC/DFC interface and thus 47S rRNA synthesis takes place here, while 5S rRNA synthesis occurs in the nucleoplasm, but is then transported to the nucleolus. Many of the early steps in pre-rRNA maturation and pre-RNP (90S and 5S RNPs) assembly occur in the DFC, with later assembly steps requiring the incorporation of additional RPs and rRNA modification by snoRNAs, occurring in the GC. The final RPs are incorporate to form the 40S and 60S subunits in the nucleoplasm, prior to the export of the individual subunits to the cytoplasm. Interestingly, the Cajal bodies have no role in direct ribosome biogenesis but provide an environment for the maturation/modification of the snRNPs (later required for splicing) and snoRNPs (later required for rRNA processing). These sub-nuclear compartments are defined by their respective protein content with the fibrillar centers (FC/DFC) containing the methyltransferase, fibrillarin, and the Cajal bodies containing coilin. It has been proposed that the exchange between these compartments is free (no active transport necessary), as no membrane exists to define or separate them. In this “free exchange model” the composition and ratio of water-soluble nucleic acids to insoluble nucleic acid binding proteins produces a semi-solid plasma state allowing for the free movement of protein–nucleic acid complexes between these compartments. Thus, it would be predicted that much of the order of these compartments is dictated by the localized protein content.

MYC a Global Regulator of Ribosome Biogenesis
It is estimated that approximately 15% of genes in higher eukaryotes contain MYC-responsive regulatory elements; thus, it is no wonder that overexpression of MYC can induce uncontrolled protein synthesis and cell proliferation. MYC is one of the only transcription factors known to regulate all three of the RNA polymerases (pol I, pol II, and pol III) and, therefore, has the capacity to induce the expression of all required rRNAs, ribosomal proteins, and co-factors necessary for ribosome biogenesis. In complex with the MAX protein, MYC binds to E-box elements upstream of the transcriptional start site in the promoter of MYC-responsive genes. Binding of the MYC:MAX heterodimer results in the recruitment of co-regulatory proteins, such as the transformation/transcription domain-associated protein (TRRAP) to the promoter. TRRAP is part of the histone acetyltransferase (HAT) complex, which is responsible for targeting acetylation of histones (H3 and H4) through the activity of the GCN5 acetyltransferase, thereby opening the DNA for transcription (Figure 1, bottom panel a,c). In the case of rDNA, MYC also results in the recruitment of RNA pol I co-factors UBF and SL-1 to the promoter, thus stimulating the transcription of the 47S pre-rRNA (Figure 1, bottom panel b). In contrast, MYC influences the transcription of the 5S rRNA and that of tRNAs in a diverse manner. Rather than forming a heterodimer with MAX, MYC associates directly with TF-IIIB in the nucleoplasm to stimulate RNA pol III-mediated transcription of theses RNAs (Figure 1, bottom panel d). Beyond the direct association with rDNA promoters, MYC is also known to influence the expression of the RNA pol I transcriptional co-factor UBP, a MYC-responsive gene product.

MYC is also known to induce the expression of both the small and large subunit ribosomal proteins, in an RNA pol II-dependent manner. Interestingly, several of these targets RPL5 and RPL11 have been found to be extremely important in sensing ribosomal stress. RPL11 has been shown not only to induce p53 transactivation, but also to bind MYC within the MYC box II domain and inhibit its association with TRRAP, thereby reducing histone acetylation and MYC-dependent transcription. Thus, the MYC-RPL11 circuit functions in a negative-feedback mode. In addition, MYC also induces a number of proteins that are either involved in rRNA processing and transport or in translation initiation. MYC controls the expression of nucleolin (NCL) and nucleophosmin (NPM), two proteins that are involved in multiple processes in the nucleus, including the processing of the 47S rRNA to 18S, 5.8S, and 28S rRNAs; as well as the expression of the nucleolar protein 56 (NOP56), a core component of the C/D box snoRNP complex, block of proliferation 1 (BOP1), part of the PeBoW complex required for 28S and 5.S rRNA maturation, and dyskerin (DKC), a H/ACA snoRNP complex subunit responsible for the pseudouridylation of rRNA species. Moreover, NPM has additional roles in the cytoplasmic-nuclear import of newly synthesized ribosomal proteins and the nuclear-cytoplasmic export of the assembling ribosomal subunits. MYC also enhances expression of the translation initiation factors eIF2α, eIF4A-I, eIF4E, and eIF4G, which regulate CAP-dependent translation and may also promote methylation of the mRNA CAP through RNA guaine-7-methytransferase.
Under certain circumstances, MYC is also known to promote apoptosis. Diverse forms of MYC can be expressed by alternate translation initiation. Two main forms, p64 and p67, result from alternate start codon usage. MYC p64 initiates from a standard AUG start codon; in contrast, MYC p67 initiates from a non-canonical upstream start codon (a CUG), which produces a protein that is 15 amino acids longer. Both these forms can associate with E-box elements, while MYC p67 can also associate with CAAT-enhancer binding elements as well, thus affecting the transcription of an additional set of genes. It has been proposed that the ration of p64 MYC to p67 MYC dictates whether MYC promotes growth/proliferation or apoptosis.

The PI3K-AKT-mTOR Pathway, Linking Ribosome Biogenesis to Extracellular Signaling
The PI3K/AKT pathway has become one of the most studied and best-characterized signal transduction pathways, due to its involvement in cell survival and proliferation, glucose metabolism, and translation. A large number of cytokine and growth factor receptors, such as the epidermal growth factor receptor (EGFR), the insulin-like growth factor receptor (IGFR), the granulocyte macrophage-colony stimulating factor receptor (GM-CSFR), and the tumor necrosis factor (TNF)-α receptors (TNFR1 and TNFR2) transduce part of their signal through the PI3K-AKT-mTOR pathway. These receptors inform the cell of the surrounding environment, whether to undergo self-renewal or differentiation. In the classical scenario, following ligand binding to its cognate receptor, the regulatory and catalytic domains of the phosphatidylinositol-3 kinase (PI3K) are recruited to the cytoplasmic domain of the receptor and activated. Activated PI3Ks catalyzes the phosphorylation of phosphatidylinositol (PtdIns), PtdIns(4)P, PtdIns(5)P, or PtdIns(4,5)P2 at the 3-position of the inositol ring to form PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 is the main form involved in AKT activation, and its level in cells is regulated by the phosphatase and tensin homolog (PTEN), the product of the mmac1 gene, which quickly dephosphorylates PtdIns (3,4,5)P3 to PtdIns(4,5)P2. It is not surprising that the gene encoding PTEN is one of the genes most often mutated or lost in cancer. PtdIns(3,4,5)P3 results in the recruitment of the AKT kinase (AKT1, -2, or -3) via the N-terminal and negative regulatory plekstrin homology (PH) domain. This association not only results in the localization of AKT to membrane components of the cell, but it also causes a conformational change in AKT, removing the negative regulation imposed by the PH domain, opening up AKT for two phosphorylation events required for its kinase activity. The phosphatidylinositol-dependent kinase, PDK1 is responsible for phosphorylating AKT1 on Thr308 (Thr309 on AKT2 and Thr308 on AKT3). Like AKT, it is recruited to the membrane via its PH domain. Phosphorylation on Thr308 is assisted by phosphorylation of Ser473 in AKT1 (Ser474 in AKT2 and Ser472 in AKT3), which is carried-out by the mTORC2 complex (Figure 2A.


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Figure 2. Diagram of the regulation and role of the PI3K-AKT-mTORC1 pathway in ribosome biogenesis and translation initiation. The diagram presents the diverse points of regulation that the PI3K-AKT-mTORC1 signaling pathway has in ribosome biogenesis and translation initiation under optimal as well as suboptimal (low ATP levels, poor nutrients, limited amino acid) conditions. 
(A) Activation of AKT through the regulation of PIP3 levels; 
(B) AKT stimulates the mTORC1 complex, which targets multiple downstream targets; 
(Cl-leucine activation of mTORC1 at the lysosome. Phosphorylation (P) marked in red represent phosphorylations that favor ribosome biogenesis and translation initiation; phosphorylations in blue represent phosphorylations that are inhibitory to ribosome biogenesis or CAP-dependent translation initiation; ubiquitinations are presented in orange.

While the AKT kinases show some redundancy in their activity, several major differences have been noticed. AKT1 and AKT2 are ubiquitously expressed, and both are present and have enzymatic roles in the nucleus and cytoplasm. In contrast, the localization of AKT3 is predominantly nuclear, with expression limited to the brain, lung, and kidney in adults; and heart, liver, and brain in fetus. AKT1 is associated more closely with anti-apoptotic/survival effects of PI3K activation, while AKT2 has been shown to be responsible for AKT-dependent insulin signaling. AKT3 is still poorly understood with few substrates identified.
Multiple substrates of AKT have been identified, but the best understood and most critical by far to ribosome biogenesis is the mTORC1 complex. The mTORC1 and mTORC2 complexes differ in several ways. First, mTORC1 serves as a substrate for AKT, while mTORC2 is responsible for the phosphorylation of AKT on Ser473. Second, mTORC1 is a rapamycin-sensitive complex while mTORC2 is not. This difference in susceptibility to rapamycin is due to the third major difference in these complexes; the protein components. Both mTORC1 and mTORC2 contain the mammalian target of rapamycin protein (mTOR), the positive/negative regulator G protein β-subunit-like (GβL or LST8), and the DEP-domain containing mTOR-interacting protein (Deptor), a negative regulator of the mTORC complexes; but while mTORC1 contains the scaffolding protein Raptor, mTORC2 contains Rictor, Sin1 (MAPKAP1), and proline-rich protein 5 (PRR5). The mTORC2 complex is nutrient insensitive, acts upstream of Rho-GTPases, and has a role in modifying the actin cytoskeleton. In contrast, the mTORC1 complex is nutrient sensitive and regulates a major part of ribosome biogenesis and CAP-dependent translation (Figure 2B).

The AKT kinases can directly phosphorylate mTOR (Thr2446, Ser2448), which increases the activity of the catalytic subunit, mTOR, but is not sufficient for mTORC1 activation. Activation of the mTORC1 complex occurs through its GTPase Rheb when in the GTP bound state. Rheb is inhibited by its GAP protein(s), the tuberous sclerosis heterodimer (TSC1/TSC2). In addition, the mTORC1 complex is also inhibited by the association of the proline-rich AKT substrate (PRAS40) in a 14-3-3 protein-dependent manner. AKT activates the mTORC1 complex by phosphorylating both PRAS40 (Thr246) and the TSC1/TSC2 (Ser939, Ser981, and Thr1462 of TSC2) complex to free Rheb and stimulate the activation of mTORC1 (Figure 2B).

Amino acid activation of mTORC1 is also possible. This involves the recruitment of mTORC1 by the Ragulator protein complex to the lysosomal membrane, following stimulation with amino acids, where mTORC1 interacts with its activator Rheb, bringing the mTORC1 complex in contact with the Rag GTPases. The heterodimeric Rag GTPases, consisting of RagA or RagB pairing with RagC or RagD, become loaded with GTP in the presence of amino acids, favoring their interaction with Raptor and the activation of mTORC1. Nicklin et al. demonstrated that it was glutamine uptake and its subsequent efflux in the presence of essential amino acids, which is the limiting step in this process. The uptake of glutamine by the cell establishes an internal reservoir of glutamine that can be exported by the heterodimeric SLC7A5-SLC3A2 antiporter. The efflux of glutamine by the antiporter promotes the import of branched-chain amino acids such as leucine. This increased presence of intracellular leucine favors the interaction of leucine with leucyl-tRNA synthetase. The leucine:leucyl-tRNA synthetase complex then acts as a GTPase-activating protein stimulating the Rag GTPases. So why leucine? Leucine happens to be the amino acid most frequently used in proteins, thus, its deficiency should set-off alarms for the cell. For this reason, it has been observed that some ribosomopathy patients can be treated with leucine supplements (see below). Moreover, the presence of Raptor in the complex assists in the recruitment of mTORC1 substrates (Figure 2C).

Finally, the activity of the mTORC1 complex can be regulated directly by the energy level of the cell. Low cellular ATP levels result in the activation of the adenosine monophosphate activated kinase (AMPK), which can phosphorylate and activate TSC2, of the TSC1–TSC2 inhibitory complex, on Thr1227 and S1345; and/or phosphorylate Raptor on Ser722 and Ser792, promoting its interaction with 14-3-3 proteins and the inhibition of mTORC1 (Figure 2). In addition, AKT has the ability to autoregulate its phosphorylation at Ser473 through the phosphorylation Sin1 (Thr86) to down-regulate mTORC2 activity.

The mTORC1 complex phosphorylates two main targets of ribosome biogenesis, the S6 kinases (p70 S6K1/p70 S6K2) and the eIF4E-binding protein (4E-BP). Recruitment of the 40S ribosomal subunit to the 5′ N7-methyl guanosine CAP [m7G(5’)ppp(5’)N] of mRNA is facilitated by the eIF4F translation initiation complex, which is composed of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the RNA helicase eIF4A. Unphosphorylated 4E-BP associates with eIF4E, blocking the association of eIF4E with eIF4G. Phosphorylation of 4E-BP, mediated by mTORC1, frees eIF4E, facilitating its association with eIF4G and the formation of the eIF4F complex; thus, favoring CAP-dependent translation. The reduced efficiency of the eIF4F complex to recognize and promote the translation of 5′ N7-methyl guanosine CAPed mRNAs favors internal ribosome entry site (IRES)-mediated translation, which is often observed during inflammation and stress. On the other hand, phosphorylation of the p70 S6 kinase, results in its activation and the subsequent downstream phosphorylation of PDCD4 (Ser67; an inhibitor of eIF4A), causing its ubiquitination and proteolysis, and eIF4B (Ser422; an activator of the eIF4A helicase), thus, favoring CAP-dependent translation of mRNAs with complex secondary structure at the 5′-end. Additionally, p70 S6K also phosphorylates polymerase delta-interacting protein 3 (POLDIP3/SKAR) on Ser383 and Ser385, favoring nuclear export and translation of spliced over non-spliced mRNAs. Under poor nutrient conditions, the eIF3 initiation complex associates with p70S6K and sequesters it in an inactive state. Following the appropriate stimulus, eIF3 is released and p70 S6K phosphorylates its targets (Figure 2B).

Beyond CAP-dependent translation initiation, the AKT-mTORC1-p70 S6 kinase pathway has been demonstrated to target other effectors of ribosome biogenesis. AKT has been shown to phosphorylate MAD1 on S145, resulting in its release from the MYC/MAX/MAD1 heterotrimer, and its degradation to form the active MYC/MAX heterodimer; thus, promoting MYC-dependent transcription. AKT also phosphorylates and stabilizes MDM2 (S166, S186, and S188), favoring the degradation of p53, a major repressor of ribosome biogenesis. In addition, AKT may regulate the type of mRNA translated based on its 5′ UTR through phosphorylation of LARP6 (S451), a protein that associates with stem-loops in the 5′UTR to stabilize mRNA. Phosphorylation of LARP6 at S451 results in LARP6 degradation. Likewise, mTOR phosphorylates LARP6 (S340, S409) and its family member LARP1 (S766, S774), but in contrast to AKT-dependent phosphorylation, mTOR-dependent phosphorylation promotes the stability and sequestering of these proteins; thus, favoring the translation of mRNAs containing the 5′terminal oligopyrimidine (TOP) motif, a 5’-cytidine followed by a short pyrimidine tract (4-14 nucleotides) immediately downstream of the methyl guanidine cap (m7Gppp) (Figure 2B). The 5’TOP mRNAs encode components of ribosome biogenesis such as the RPS and RPL proteins. Moreover, mTORC1 is able to stimulate RNA pol I-dependent transcription of the 47S rRNA by activation of UBF and TIF-1A; and RNA pol III-dependent transcription of 5S rRNA and tRNA through its direct recruitment to the promoter, by TF-IIIB, and subsequent phosphorylation of MAF1 (S60, S68, and S75), an inhibitor of the TF-IIIB complex formation; thus, establishing a role for mTORC1 as a transcription factor. Use of the mTOR inhibitor rapamycin blocks the synthesis of rDNA by inhibiting the formation of the RNA pol I and RNA pol III transcription complexes on their respective promoters. It is also apparent that mTOR may also regulate the balance between mTORC1 and mTORC2 complex formation by phosphorylating diverse components of the mTORC complexes, including itself. Finally, the p70 S6 kinases also phosphorylate RPS6 and the eukaryotic elongation factor 2α (eEF2α) kinase (eEF2K). Phosphorylation of RPS6 results in the enhanced translation of 5′TOP RNAs, while phosphorylation of eEF2K results in inhibition of its catalytic activity and the activation of eEF2α, favoring translation elongation. RPS6 can also be phosphorylated by p90RSK, which is activated downstream of the RAS-RAF-ERK pathway activation. In addition to these, p70 S6K appears to also regulate AKT and mTORC1 activity through phosphorylation of Thr2446 and Ser2448 of mTOR and the phosphorylation and targeted degradation of Deptor (Ser286, Ser287, and Ser291) and Rictor (Thr1135). The phosphorylation of Rictor is considered a negative feedback modification as it results in decreased mTORC2 phosphorylation of AKT.

Recently, Bavelloni et al. reported a study in which they sought to identify novel nuclear AKT substrates. Using phospho-AKT substrate specific antibodies coupled with mass spectrometery analysis, the authors identified a set of proteins present in the nuclear lysates of two hematopoietic cell lines that were immunoprecipitated with antibodies recognizing the following epitopes: K/R-x-K/R-x-x-S*/T* or R-x-x-S*/T*; where “x” represents any amino acid and the asterisk represents a phosphorylated amino acid. The authors then analyzed the identified proteins to determine if they actually contained sites that could be recognized by the antibodies employed in the study. Both AKT and p70 S6K belong to the AGC kinase family and have similar phosphorylation consensus sites; thus, the identified proteins may represent both AKT and p70 S6K substrates (Table 1). Many of the identified proteins are intimately related to ribosome biogenesis. Thus, it is possible AKT-mTOR-p70 S6K signaling has additional targets that influence ribosome biogenesis and translation initiation that have yet to be characterized.

Cell Cycle Regulators and Ribosomal Stress

Coordination between cell division and proteins synthesis is imperative for cell survival; thus, it is not surprising that multiple regulators of the cell cycle also have a significant role in controlling ribosome biogenesis. Unphosphorylated retinoblastoma protein (Rb) family members not only regulate the cell cycle by associating with the E2F transcription factor, but their hypophosphorylated forms also directly associate with UBF of the RNA pol I complex and TF-IIIB of the RNA pol III complex, inhibiting the synthesis of the 47S and 5S rRNAs, as well as the necessary snoRNAs and tRNAs (Figure 3A).


Translation through ribosomes,  amazing nano machines - Page 2 Regula10

Figure 3. Regulation of ribosome biogenesis by cell cycle regulators. (A) The cell cycle regulators/tumor suppressors pRb/p130 family and p53 regulate ribosome biogenesis by suppressing rDNA transcription initiation. (B) The tumor suppressor p14ARF, an alternate open reading frame of the p16INK4A gene, regulates ribosome biogenesis and translation initiation through its interaction with NPM1 and MDM2, resulting in p53 stability and transactivation and eIF2α phosphorylation. (C) Faulty assembly of ribosomal proteins (RPS and RPL) result in elevated levels of the pre-assembly RNP complex 5S RNP (RPL5-RPL11-5S rRNA), which binds to MDM2 resulting in p53 stability and transactivation. Additional RPSs and RPLs have been reported to bind MDM2 as well, resulting in the same effects on p53.

Loss of Rb expression or hyperphosphorylation of Rb, due to the activation of the cyclin-dependent kinase (CDK)-cyclin complex, results in the removal of this level of control. Additionally, the smaller of the products of the ink4a tumor suppressor gene, p14ARF (p19ARF in mice) also associates with proteins of the RNA pol I complex affecting both 47S rRNA transcriptional initiation and termination. More interestingly, p14ARF has a significant role in regulating both rRNA processing, as well as p53-dependent transcription through its association with nucleophosmin (NPM1). NPM1 is a highly expressed nuclear phosphoprotein involved in diverse cellular processes (rRNA processing, ribosome protein nuclear import, ribosome assembly, and ribosome subunit nuclear export). NPM1 associates with diverse proteins, influencing their activity; among these are the p53 ubiquitinase MDM2 and the dsRNA-dependent inflammatory/stress activated kinase, PKR. When bound to NPM, these proteins are sequestered to the nucleus. Enhanced expression of p14ARF results in its association with NPM and the formation of an MDM2 inhibitory complex, thus stabilizing p53. Similarly, the sequestration of PKR, by NPM, keeps it localized to the nucleus where its localization is associated with cell growth and DNA repair (Figure 3B). Garcia et al. reported that, following viral infection, enhanced expression of p14ARF promoted its association with NPM, resulting in the release of PKR and the translocation of PKR to the cytoplasm, where it phosphorylates eIF2α, resulting in the inhibition of protein synthesis .

The p53 transcription factor is a master regulator of the cell. Most of the genes induced by p53 are involved in cell cycle regulation (arrest) and apoptosis; therefore, stimulation of p53 leads to cell cycle arrest and repair or subsequent cell death in most cases. In addition, p53 shares the stage with two closely related family members, p63 and p73. These family members may cooperate with or antagonize one another, depending on the promoter and the gene in question. Approximately 50% of human tumors contain mutant p53. These mutations are known to affect the transactivation capacity of p53, p53 stability, and the ability of p53 to interact with additional cofactors.

The status of p53 is also extremely important in the regulation of ribosome biogenesis. The association of p53 with SL-1 complex of RNA pol I or TF-IIIB of the RNA pol III complex results in transcriptional repression of these rRNA, tRNA, and snoRNA genes. The interaction of p53 with RNA pol II-dependent promoters can either stimulate or repress their transcription. As stated above, p53 protein levels are chiefly regulated at the level of protein stability. The E3 ubiquitin ligase MDM2 associates with p53 and ubiquitinates it, thereby targeting it for degradation by the proteosome. Interestingly, p53 binding of the mdm2 gene stimulates the synthesis of its transcript, thus p53 can autoregulate its expression through the induction of MDM2. The mechanisms that regulate ribosome biogenesis have taken advantage of the MDM2-p53 relationship as a checkpoint for ribosomal stress. Alterations in the levels of proteins required for rRNA synthesis, processing, and transport can influence the MDM2-p53 interaction. Thus, alterations that may impinge on the early steps of ribosomal biogenesis also influence p53 stability, favoring the accumulation of p53. Among these proteins are NPM1 (see above) and nucleostemin (NS). Overexpression of NS causes its accumulation in the nucleoplasm and its association with MDM2 via interaction of the coiled–coiled domains of NS with the acidic domain of MDM2, thus inhibiting p53 ubiquitination and enhancing p53 stability. In contrast, depletion of NS activates p53 through the ribosomal protein pathway.
Additionally, the accumulation of rRNAs must match the level of rRNA processing protein complexes, which must match the synthesis of ribosomal proteins to be incorporated into the assembling ribosome, which must match the transport/chaperone proteins available. A certain amount of leeway must be inherent in the system and controlled or “tweaked” through transient activation stimulation of key signal transduction pathways. The system must also have the ability to alter the assembly of the ribosome to favor the translation of certain mRNAs over others when necessary for the cell. The obvious disproportion of the necessary RNA or protein intermediates results in the stimulation of p53 transcriptional activity, arresting the process of ribosome biogenesis. This safety switch for the organism impedes the production of ribosomes that possess altered activity, which could be deleterious to the organism (constitutive p53 activity can also contribute to disease—see below). Several ribosomal proteins are known to bind to MDM2 and inhibit its activity toward p53; these include RPS3, RPS7, RPL5, RPL11, and RPL23 (Figure 3C).

The ribosome proteins are produced in excess in the cytoplasm. The stability of these proteins is dependent on their interaction with chaperones and nuclear import proteins. Those RPs not associated with chaperones and directed to the nucleus for ribosome assembly are quickly degraded by the ubiquitin proteosome complex (UPC). Thus, free nucleolar/nucleoplasm accumulation of these RPs would signal a failure in the maturation process of the 40S and 60S subunits and stimulate p53. One of the more interesting complexes is the RPL5-RPL11-5S rRNA (5S RNP). This complex, which has an early and critical role in rRNA processing, is an early sentinel for defects in ribosome biogenesis.  RPL5-RPL11-5S rRNA accumulation and association with MDM2 could take place in both the nucleolus and nucleoplasm . Interestingly, the RPL5-RPL11-5SRNP complex is also likely responsible for p53 accumulation in response to the deficiency of many of the additional ribosomal proteins that do not directly interact with MDM2 (Figure 3C).

EIF2α Regulation and Translation Initiation: The PKR Story

The ultimate goal of ribosome biogenesis is to produce ribosomes capable of accurately and successfully translating mRNAs into protein. Like ribosome biogenesis, the process of translation has a rate-limiting step, which is initiation; therefore, in addition to the eIF4F translation initiation complex, which is under the control of the AKT-mTOR and RAS-RAF-MAPK pathways, two additional initiation factors represent major points of translation control, eIF2 and eIF2B. These complexes bare both the GTP and the Met-tRNA necessary for pre-initiation complex (PIC) formation and translation initiation, as well as the proteins for the GDP to GTP exchange required to initiate the next round of translation. Regulation of eIF2 is via the α-subunit (eIF2α) and is probably the best understood mechanism regulating translation initiation. One of four different kinases (PKR, PERK, GCN2, or HRI) leads to the phosphorylation of eIF2α. Phosphorylation of eIF2α on Ser51 results in eIF2 being locked in the GDP bound state with eIF2B, unable to catalyze the initiation of protein synthesis. As the eIF2 complex is limited compared to eIF2B, it does not take much phosphorylated eIF2α to soon result in a complete block of general translation. Although each of these kinases phosphorylates eIF2α on the same serine residue, they do so in response to differing stresses. The PKR-like endoplasmic reticulum kinase (PERK) is mainly activated following ER stress, as part of the unfolded protein response (UPR) and has been shown in mice to be inhibited by AKT1-dependent phosphorylation. The general control nonderepressable-2 (GCN2) is part of the nutrient sensing pathway and responds to amino acid starvation; lack of amino acids results in uncharged tRNAs, which stimulate GCN2 kinase activation. Heme-regulated eukaryotic initiation factor-2-alpha kinase (HRI), which is expressed mainly in cells of erythroid lineage and the first of the eIF2α kinases to be identified, is activated in response to low heme concentrations. The double-strand RNA-dependent kinase PKR, on the other hand, is activated in response to the most diverse types of stresses; among these are: viral infection, dsRNA, peroxidation, mitochondrial stress, DNA damage, ER stress, inflammatory cytokines, growth factor deprivation, and Toll-like receptor activation. Together, these kinases form a network that can regulate translation initiation under a myriad of stress conditions. Interestingly, while each of these kinases can be found in the cytoplasm, PKR is the only eIF2α kinase that is also present in the nucleolus and nucleoplasm.
From prokaryotes to mammals, ribosome biogenesis and subsequent translation are highly regulated by the surrounding environment to limit energy expenditure under conditions that are unfavorable for growth, as well as limit the possibility of producing mutant proteins. While phosphorylation of eIF2α was long thought to be strictly pro-apoptotic, this is not the case. Phosphorylation of eIF2α results in a shut-down of general CAP-dependent translation but, at the same time, it allows for efficient translation of upstream open reading frames (uORFs) in particular mRNAs that contain complex secondary structure at the 5’ end and an IRES element upstream . Short-term inhibition of general translation through eIF2α phosphorylation establishes a pro-survival state by allowing for cellular repair and time for the cell to adjust following a particular stress. If this stress cannot be resolved and general translation remains inhibited, the cell will likely die through apoptotic means; thus, a coordinated interaction between the eIF2α kinases and the AKT-mTOR-p70 S6K pathway must be present. In contrast, under other conditions, the phosphorylation of eIF2α has been shown to inhibit IRES-mediated translation. These differences may be due, in part, to the presence of specific regulator proteins that differ between IRES elements and are, therefore, specific to the being mRNAs translated (Figure 4).

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Figure 4. PKR regulates both translation initiation and ribosome biogenesis. The diverse points that PKR regulates in ribosome biogenesis and translation initiation are indicated. PKR regulates general CAP-dependent translation initiation and favors alternative translation initiation (ex., IRES) through the phosphorylation of eIFα or the modulation of GSK3α/β phosphorylation by phosphatases. Regulation of ribosome biogenesis is through PKR-mediated effects on p53 stability and MYC isoform expression. PKR likely also influences a number of additional proteins critical to ribosome biogenesis with which it interacts in the nucleus, including diverse ribosomal proteins. Phosphorylation (P) marked in red represent phosphorylations that enhance the activity of the recipient protein; phosphorylations in blue represent phosphorylations that are inhibitory to the recipient protein.




New twist to nuclear import
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738017/

https://www.youtube.com/watch?v=-ROJOBDCCLE&feature=youtu.be
You have to be able to synthesize the peptide bond okay but that doesn't solve the problem you also have to do it over and over again in order to make something useful in size


1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1171055/pdf/007078.pdf



Last edited by Otangelo on Wed Mar 10, 2021 12:35 pm; edited 25 times in total

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Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number 13 February 2018

Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. 

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA non-coding RNA molecules, typically 20-27 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway.
https://en.wikipedia.org/wiki/Small_interfering_RNA

We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription, pre-ribosomal RNA (pre-rRNA) processing, and/or global protein synthesis. This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

Ribosome biogenesis is a highly regulated cellular process essential for growth and development. In humans, production of ribosomes begins in the cell nucleolus with the transcription of a 47S precursor rRNA (pre-rRNA) by RNA polymerase I (RNAPI). This 47S pre-rRNA is transcribed from the 5 acrocentric chromosomes in humans that bear the repeated rDNA sequences. The 47S pre-rRNA is chemically modified and processed before assembly with the 5S rRNA into the mature ribosomes that are essential for protein synthesis. Production of a single human ribosome requires over 200 assembly factors, 80 ribosomal proteins (r-proteins), and all three RNA polymerases and takes place in the nucleolus, nucleus, and cytoplasm of cells. This process is subject to complex regulation because it must be highly responsive to various cellular stimuli, such as nutrient availability. Ribosome biogenesis in human cells has great complexity in regulation.

Ribosome biogenesis is a complex and essential process that must be performed accurately and often. Therefore, human cells must be able to effectively coordinate ribosome biogenesis with a wide range of cellular cues. This screen highlights how ribosome biogenesis is enmeshed in such diverse cellular processes. Further exploration of the crosstalk between ribosome production and diverse non-nucleolar processes is essential to understanding the link between the nucleolus and human disease.

1. https://www.sciencedirect.com/science/article/pii/S2211124718301050

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The ribosome as a missing link in the evolution of life 21 February 2015

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

Many steps in the evolution origin of cellular life are still mysterious. 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 origin of cells. We present evidence that the entire set of transfer RNAs for all twenty amino acids are encoded in both the 16S and 23S rRNAs of Escherichia coli K12; that nucleotide sequences that could encode key fragments of ribosomal proteins, polymerases, ligases, synthetases, and phosphatases are to be found in each of the six possible reading frames of the 16S and 23S rRNAs; and that every sequence of bases in rRNA has information encoding more than one of these functions in addition to acting as a structural component of the ribosome. Ribosomal RNA, in short, is not just a structural scaffold for proteins, but the vestigial remnant of a primordial genome that may have encoded a self-organizing, self-replicating, auto-catalytic intermediary between macromolecules and cellular life.

Translation through ribosomes,  amazing nano machines - Page 2 1-s2_011

A difficulty in accounting for the emergence of life is to explain how something as complex as a living cell could  evolve  emerge. At present, several general approaches dominate evolutionary thinking. Working from simplicity to complexity, RNA-world, or “genetics-first” models and compositional replication, or “metabolism-first” models together provide insights into early prebiotic evolution origin from simple molecules to the first polymers and polymer aggregates. Neither of these types of models fully explains the evolution origin of cells. RNA-world models cannot explain the evolution of metabolism and generally fail to take into account the fact that amino acids (and therefore peptides and proteins) almost certainly were synthesized along with polynucleotides under prebiotic conditions, making it almost certain that these classes of molecules co-evolved 

My comment:  "Making it almost certain" based on what evidence? There is none. This is an entirely made-up assertion that is not backed up by evidence. Also, the authors constantly attempt to smuggle evolution as a concept/mechanism into their narrative, but prebiotic evolution is a contradiction in terms. There was no evolution prior to DNA replication.

Compositional replication models can explain such co-evolution

How did Metabolism and Genetic Replication Get Married?  14 October 2012
https://sci-hub.ren/10.1007/s11084-012-9312-3
We propose the Accordion model in which a dynamic interface between lipid domains catalysed monomer to polymer reactions and became decorated with peptides and nucleotides that favoured their own catalysis. Starting with a prebiotic ecology of molecules some of which bound to one another with different affinities, it has been proposed that non-covalent assemblies, protocells or composomes grew in a flux of abiotic creation and destruction because their constituent molecules were preserved by their interactions with one another. Fission-fusion between composomes may then have led to a selection for stability from which coding emerged.  In this scenario, composomes were structured and lipid domains with interfaces that catalysed that formation of oligonucleotides and peptides. The most fundamental of these constraints was–and remains–that of compromising the need to grow with the need to survive. These very different behaviours require two very different types of structure: non-equilibrium structures (for growth) and equilibrium structures (for survival). I

My comment: Above paper is a prime example sciency mambo-Jambo, just so stories, pseudo-science, and unwarranted speculation. Sad, that so many take such ink as serious science, while it is just bad science, which cannot be taken seriously.

but not how linear replication schemes became dominant. Moreover, neither type of model accounts for how simple replicable molecules or aggregates of molecules evolved into complex cells with organized compartments and structures such as ribosomes, acidocalcisomes, and functional membranes that incorporated specialized transporters and receptors. Models of the last universal common ancestor (LUCA) – the presumptive first cellular form of life  – attempt to resolve some of these problems by working from complexity toward simplicity. LUCA models provide insight (with much disagreement) into the minimum complexity required for cellularity but reveal little about the preceding evolutionary steps. The gap is enormous between the simplicity-toward-complexity models, which can suggest how simple replication of small sets of polymers may have emerged, and complexity-toward-simplicity models, which suggest a minimum of several hundred genes and their products networked within specialized metabolic compartments. What kind of evolvable entities might bridge this gap?

Evolvable entities existing between self-replicating polymers and fully functional cells would presumably have many, though not all, of the functions of a cell, yet be significantly simpler in composition and organization. These entities would be able to self-organize and replicate themselves; store information and replicate that information; translate the information into the components necessary to produce their functional structures; capture metabolic components and energy; and transform these into useful biochemical networks. 

We suggest that a ribosome-like entity was one of the key intermediaries between prebiotic and cellular evolution.

Ribosomes are prerequisites to all cellular life, ubiquitously conserved, with genetic roots that pre-date LUCA, and therefore entities that had to evolve emerge prior to cellular life itself. While the ribosome may not be capable of the broad metabolic processes that characterize cellular life, the ribosome is a self-organizing complex composed of both polynucleotides and proteins that could link RNA-world to compositional replication concepts in the origins of life. Moreover, ribosomes carry out some of the most fundamental processes characteristic of living systems, including a coordinated series of chemical reactions capable of translating genetic information into functional proteins. What ribosomes are not thought to do is to carry genetic information, and in particular the genetic information required to encode their own structures and functions. 

But what if ribosomal RNA (rRNA), which is generally considered to be simply a structural component of ribosomes, actually represents a primitive genome encoding the genetic information needed to direct ribosomal replication, translation and self-organization?

Respose:  RNA could not replace DNA, because it is too unstable.

An intermediate hypothesis might be that the amount of genetic information encoded in rRNA is purely random and therefore the number of tRNAs and ribosome-related proteins that rRNA encodes will be no more or less than any random assortment of any other set of randomly chosen RNAs.

My comment: Purely random? Are the authors OUT OF THEIR SANE MIND? This is simply unwarranted speculation, there is no evidence that permits to point to that possibility. 

“Selfish” ribosomes, in short, provide one potential intermediary in the process of evolution from the first macromolecules to hyperstructures and finally cells.

My comment: The authors have gotten just one thing right. Ribosomes had to emerge prior to cellular life. All other assertions are unwarranted, in short, nonsensical speculation.


1. https://www.sciencedirect.com/science/article/pii/S0022519314006778?via%3Dihub

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Machines, that make machines, that make machines, that make subunits of more complex machines: By evolution, or design?

https://reasonandscience.catsboard.com/t2061p225-my-articles#8471

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. If, on top of that, the entire assembly process would have to be without external input of information, nor intelligent action or direction, that would be an entirely new level of sophistication required. 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 in a fully autonomous way. The entire manufacturing process would have to be self-regulated, and under constant assembly control. The environment of the factories would have to be constantly monitored, and any change, like temperature, pH, air purity etc. detected and maintained in an acceptable level, and any deviation, corrected.  It is evident, that the implementation of such a complex manufacturing process requires foresight and foreknowledge , and know how.  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, the protein factory.  

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 (scaRNAs), 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.

A great machine, subdivided into dozens of smaller machines, that contribute to the higher order and function of the greater machine, everything assembled to work in a cooperative interlocked way, in a joint venture, requires foresight of how to  assemble the whole machine.

David Hume: All these various machines, and even their most minute parts, are adjusted to each other with an accuracy which ravishes into admiration all men who have ever contemplated them.

History of the ribosome and the origin of translation  November 30, 2015
The ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA.

My comment: How does that make sense? It doesn't. This is a prime example of bad science, a peer reviewed science paper, that supposedly merits credit. No. Sadly. It doesn't.

Translation through ribosomes,  amazing nano machines - Page 2 5cb7e7b6e6f27f6a88e9d68994ce4000

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