Translation through ribosomes, amazing nano machines

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 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).


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).



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).


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


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


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.


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). 



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

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. 



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. 


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. 



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. 


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


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)





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

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.

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
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), 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.
A 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 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. 

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 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.











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.
A 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, complex, finely 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. 

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). 


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).





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.






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; 
(C) l-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).




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).

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).


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

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

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.



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

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.

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

Ribosomes are large multimolecular machines that synthesize proteins from amino acids in living cells.The process of making a single eukaryotic ribosome is a herculean task. Eukaryotes have 80S ribosomes  consisting of a small (40S) and large (60S) subunit. Their 40S subunit has an 18S RNA (1900 nucleotides) and 33 proteins. The large subunit is composed of a 5S RNA (120 nucleotides), 28S RNA (4700 nucleotides), a 5.8S RNA (160 nucleotides) subunits and 49 proteins.

In Saccharomyces cerevisiae (single-celled fungus microorganisms), nearly 7000 nucleotides of pre-rRNA must be accurately transcribed, cleaved, folded, chemically modified by 71 snoRNPs  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 cell) 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) and facilitate cleavage of rRNA precursors. They contain a component called snoRNA. Small nucleolar RNAs (snoRNAs) are a class of small RNA  molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs , transfer RNAs  and small nuclear RNAs .

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

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

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

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

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

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

Biosynthesis, assembly, and transport of sno/scaRNPs 3
The 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
Nearly all sno/scaRNAs are intronic sequences that are freed from the primary transcript by endonucleases or by splicing after mRNA processing. Protein binding near sno/scaRNA terminals trigger exonucleases to degrade both ends of the intronic sequence until reaching the sno/scaRNA structure, where further degradation is inhibited by a bound protein and the mature sno/scaRNA is released

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

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

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. 

Syllogisms about the Ribosome

https://reasonandscience.catsboard.com/t1661p25-translation-through-ribosomes-amazing-nano-machines#8800

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

Evidence of design in the ribosome
1. Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. For example, the functional group A2451, one single ribonucleotide which is not only of vital importance for peptide bond catalysis ( yeah, how did unguided events "discover" hot to place this one tiny molecule in the right place amongst 2900 of its peers ) but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here" !! and the ribosome takes action. Ribosomes form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, they can halt the translation process on the fly, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential. Even more strikingly amazing, ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for instance, for a messenger RNA that has broken and is thus is missing its stop codon. The ribosome uses an incredible piece of bio-engineering to rescue a stalled situation when they get stuck at the end of the truncated chain. They use a mechanism called Trans-translation. It is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. This mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. This so-called Trans-Translation is performed by a ribonucleoprotein complex.
2. The set up of a language, and upon it, the programming of a completely autonomous communication network, which directs the operation of a complex factory, which during operation error checks and performs repairs, to make specific purposeful products, is always the product of an intelligent agency.
3. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confesses that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: an intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

Evidence of design in the ribosome
1. Ribosomes have the purpose to translate genetic information into proteins. According to Craig Venter, the ribosome is “an incredibly beautiful complex entity” which requires a minimum of 53 proteins. It is nothing if not an editorial perfectionist…the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products…  They are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They can halt the translation process on the fly, and coordinate extremely complex movements. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.
2. For the assembly of this protein-making factory, consisting of multiple parts, the following is required: genetic information to produce the ribosome assembly proteins, chaperones, all ribosome subunits, and assembly cofactors. a full set of tRNA's, a full set of aminoacyl tRNA synthetases, the signal recognition particle, elongation factors, mRNA, etc. The individual parts must be available,  precisely fit together, and assembly must be coordinated. A ribosome cannot perform its function unless all subparts are fully set up and interlocked.
3. The making of a translation machine makes only sense if there is a source code, and information to be translated. Eugene Koonin: Breaking the evolution of the translation system into incremental steps, each associated with a biologically plausible selective advantage is extremely difficult even within a speculative scheme let alone experimentally. Speaking of ribosomes, they are so well structured that when broken down into their component parts by chemical catalysts (into long molecular fragments and more than fifty different proteins) they reform into a functioning ribosome as soon as the divisive chemical forces have been removed, independent of any enzymes or assembly machinery – and carry on working.  Design some machinery which behaves like this and I personally will build a temple to your name! Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. The origin of the ribosome is better explained through a brilliant intelligent and powerful designer, rather than mindless natural processes by chance, or/and evolution since we observe all the time minds capabilities producing machines and factories.

Translation through the ribosome is an irreducible, integrated complex process
1. The ribosome is the 3D printer of proteins. A human-made 3D printer is made of several functional parts, like the nozzle, the extruder, cooling fan, heated be, the painter's tape, etc. The 3D extruder has no use on its own. But only, when working inside the 3D printer in the right place. A bacterial cell depends upon a translation and coding system consisting of 106 distinct but functionally integrated proteins as well several distinct types of RNA molecules (tRNAs, mRNAs, and rRNAs). This system includes the ribosome (consisting of fifty distinct protein parts), the twenty distinct tRNA synthetases, twenty distinct tRNA molecules with their specific anticodons, about 200 ribosome assembly proteins and 75 co-factors, chaperones, free-floating amino acids, ATP molecules (for energy), and—last, but not least—information-rich mRNA transcripts for directing protein synthesis. Many of the proteins in the translation system perform multiple functions and catalyze coordinated multistep chemical transformations.
2. In the same sense, as an engineer would not project, invent, create and make a blueprint of a 3D printer extruder with no use by its own, but only conjoined, and together with all other parts while projecting a whole printer, envisioning its end function and use, its evident that unguided random natural events without foresight would not come up with an assemblage of tiny molecular machines, enzymatic structures with unique contours, which bear no function by their own, but only when inserted as part of the ribosome with higher ends, being essential for cells to translate DNA information into proteins, and being a key part participating to perpetuate life. Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. In other words: Why would natural selection select an intermediate biosynthesis product, which has by its own no use for the organism, unless that product keeps going through all necessary steps, up to the point to be ready to be assembled in a larger system?  Never do we see blind, unguided processes leading to complex functional systems with integrated parts contributing to the overarching design goal. A minimal amount of instructional complex information is required for a gene to produce useful proteins. A minimal size of a protein is necessary for it to be functional.   Thus, before a region of DNA contains the requisite information to make useful proteins, natural selection would not select for a positive trait and play no role in guiding its evolution.
3. Naturalistic mechanisms or undirected causes do not suffice to explain the origin of information (instructed complex information), irreducible complexity, and the setup of complex machines with little tolerance of change.   Therefore, intelligent design constitutes the best explanations for the origin of the information guiding the making of the irreducible and integrated complex ribosome protein factory.

Evidence of design in the ribosome
1.  Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. For example, the functional group A2451, one single ribonucleotide which is not only of vital importance for peptide bond catalysis ( yeah, how did unguided events "discover" hot to place this one tiny molecule in the right place amongst 2900 of its peers ) but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here" !! and the ribosome takes action. Ribosomes form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, they can halt the translation process on the fly, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential. Even more strikingly, ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for instance, for a messenger RNA that has broken and is thus is missing its stop codon. The ribosome uses an incredible piece of bio-engineering to rescue a stalled situation when they get stuck at the end of the truncated chain. They use a mechanism called Trans-translation. It is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. This mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. This so-called Trans-Translation is performed by a ribonucleoprotein complex.
2. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confesses that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation:
3. An intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

The adapter key machinery to translate genetic Information is irreducibly complex
1. Ribosomes have the purpose to translate genetic information into proteins. According to Craig Venter, the ribosome is “an incredibly beautiful complex entity” which requires a minimum of 53 proteins. It is nothing if not an editorial perfectionist…the ribosome exerts far tighter quality control than anyone ever suspected over its precious protein products…  They are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. Furthermore, they communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They can halt the translation process on the fly, and coordinate extremely complex movements. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential.
2. For the assembly of this protein making factory, consisting of multiple parts, the following is required: genetic information to produce the ribosome assembly proteins, chaperones, all ribosome subunits and assembly cofactors. A full set of tRNA's, a full set of aminoacyl tRNA synthetases, the signal recognition particle, elongation factors, mRNA, etc. The individual parts must be available,  precisely fit together, and assembly must be coordinated. A ribosome cannot perform its function unless all subparts are fully set up and interlocked.
3. The making of a translation machine makes only sense if there is a source code, and information to be translated. Eugene Koonin: Breaking the evolution of the translation system into incremental steps, each associated with a biologically plausible selective advantage is extremely difficult even within a speculative scheme let alone experimentally. Speaking of ribosomes, they are so well-structured that when broken down into their component parts by chemical catalysts (into long molecular fragments and more than fifty different proteins) they reform into a functioning ribosome as soon as the divisive chemical forces have been removed, independent of any enzymes or assembly machinery – and carry on working.  Design some machinery which behaves like this, and I personally will build a temple to your name! Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system. The origin of the ribosome is better explained through a brilliant intelligent and powerful designer, rather than mindless natural processes by chance, or/and evolution since we observe all the time minds capabilities producing machines and factories.

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

Evidence of design in the ribosome
1.  Ribosomes are molecular factories with complex machine-like operations. They carefully sense, transfer, and process, continually exchange and integrate information during the various steps of translation, within itself at a molecular scale, and amazingly, even make decisions. For example, the functional group A2451, one single ribonucleotide which is not only of vital importance for peptide bond catalysis ( yeah, how did unguided events "discover" hot to place this one tiny molecule in the right place amongst 2900 of its peers ) but when the translation process is stalled, it signals to a dynamically coupled group in the exit tunnel of the product, the polypeptide chain: " we have a problem here" !! and the ribosome takes action. Ribosomes form complex circuits. They perform masterfully long-range signaling and perform information transfer between remote functional sites. They communicate in a coordinated manner, and information is integrated and processed to enable an optimized ribosome activity. Strikingly, many of the ribosome functional properties go far beyond the skills of a simple mechanical machine. They choreograph, collaborate, modulate, regulate, monitor the translation status, sensor quality, synchronize, they can halt the translation process on the fly, and coordinate extremely complex movements, like rotations and elongations, even helped by external synchronization systems. to direct movements during translation. The whole system incorporates 11 ingenious error check and repair mechanisms, to guarantee faithful and accurate translation, which is life-essential. Even more strikingly, ribosomes occasionally get stalled when faulty messenger RNA molecules are read, for instance, for a messenger RNA that has broken and is thus is missing its stop codon. The ribosome uses an incredible piece of bio-engineering to rescue a stalled situation when they get stuck at the end of the truncated chain. They use a mechanism called Trans-translation. It is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. This mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. This so-called Trans-Translation is performed by a ribonucleoprotein complex.
2. The Ribosome had to be fully operational when life began. This means the origin of the Ribosome cannot be explained by Darwinian evolution. No wonder does science confesses that the history of these polypeptides remains an enigma. But for us, theists, the enigma has an explanation: 
3. An intelligent cognitive agency, a powerful creator, God, through his direct intervention, wonderful creative force, and activity, created this awe-inspiring life-essential factory inside of many orders of magnitude greater cell factories, fully operational right from the beginning.

Error detection and repair during the biogenesis & maturation of the ribosome, tRNA's, Aminoacyl-tRNA synthetases, and translation: by chance, or design?
1. In cells, in a variety of biochemical processes, when something goes havoc for some reason,  there is readily an armada of different error check and repair mechanisms with their "antenna" out to detect errors, and correct them, preventing lethal consequences.
2. Leaking Cells membranes need to be fixed. During DNA replication, and translation, error check and repair is essential. Cells are endowed with  a wide variety of specialized DNA repair mechanisms to counteract daily attacks: base excision repair, nucleotide excision repair, homologous recombination repair, mismatch repair, photoreactivation, nonhomologous end joining, translesion synthesis, and processing by the MRN complex. The Ribosome alone has 13 different error-check and repair mechanisms.  In addition to repairing damage to existing DNA, living organisms have mechanisms to correct errors during reproduction. Bacteria have three types of DNA polymerase, all capable of detecting an incorrect base pairing, backing up one step to excise the incorrect nucleotide, and then progressing forward in a process called proofreading. The proofreading step decreases the error rate in bacteria from approximately one error in 100,000 base pairs to one error in 10,000,000 base pairs.
3. Molecules don't care if they are assembled in a way to bear a specific function. And if they do and the function is damaged and breaks down, those molecules neither "care" that they cease bearing that function. 
4. Know how to implement an error check and repair system requires foresight. The very concepts of proofreading and repair implies goal orientation and "know-how" to keep something working and going. Those things can only come from an intelligent agency which implements these systems for specific purposes.

Protein synthesis is of central importance for cells, and they need to be synthesized at the right time when cells need them, in order to be apt to adapt to changing environments. Bacterial cells contain [up] to 100,000 ribosomes.  
During translation, messenger RNA enters the ribosome, containing a sequence of codons, genetic words, that have to be translated. Each codon word is assigned to an amino acid, There are 20 different tRNAs ( to which amino acids are attached) randomly colliding with the ribosome and the correct one needs to be chosen from those, that corresponds to the codon. This recognition process occurs in less than 0.1 seconds. That is including error check and eventual exclusion if a wrong tRNA is added. And the bond formation of one amino acid to another to form the polypeptide chain occurs at an incredible speed, namely up to 20 peptide bonds per second. It is as if an assembly-line worker would need to choose the correct part from a stream of randomly arrayed parts and then assemble it before the next part could be added. What makes the biological systems remarkable is that they have the "know-how" inbuilt to rapidly determine if the randomly sampled part is the correct one before proceeding.

Question: How could that ribonucleotide machine have ever emerged randomly, and then started its operation at such incredible speeds, efficiency, robustness, including error check and repair mechanisms operating to maintain high fidelity during protein synthesis?  How could or would the ribosome ever emerge by incremental steps, where an intermediate step would be nonfunctional? Atheists can accuse me of being incredulous that it could happen by chance. And they are right. I am.

The ribosome is one of the largest and most complex macromolecular machines in living cells. It has functions more complex than virtually any other enzyme. It was required and indispensable for life to start. How does that fit the evolutionary narrative, from simple, to complex? It is a paradox - unless we perform a paradigm shift and substitute the evolutionary narrative with creation.

Alexey Rozov: Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction 07 June 2019
The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.
https://www.nature.com/articles/s41467-019-10409-4

Michael T. Englander: The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center April 27, 2015
https://www.pnas.org/doi/10.1073/pnas.1424712112

Alexey Rozov: Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction  2019 Jun 7
Metal ions play key roles in a broad range of cellular processes. Typically, alkali and alkaline earth metals, such as Na+, K+, Mg2+, Ca2+ are critical for the stability, proper folding and functioning of RNA and proteins, whereas transition metals are also involved in catalysis of redox reactions (Fe2+ or Cu2+)8,9 or act as Lewis acids in enzyme active sites (Zn2+). In the specific case of RNA, cations facilitate the dense folding arrangements of the negatively charged phosphate backbone of RNA molecules while enabling various cellular functions: gene expres​sion(messenger (m-) RNAs and transfer (t-) RNAs), gene regulation (small nuclear, micro and small interfering RNAs), enzymatic activity (e.g., ribozymes), or resistance to pathogenic and parasitic invaders, observed in Eukarya. Ribosomes perform protein synthesis upon subunit association and interaction with mRNA and tRNA ligands; their structure and function strongly depend on the presence of divalent (mainly Mg2+, Zn2+) and monovalent (mainly K+, NH4 +) cations. Magnesium is the most characterized cation, its importance for ribosome activity was described by pioneers of ribosome research. Lack of Mg2+ in growth medium for E. coli induces ribosome degradation.  In vitro studies demonstrated that Mg2+ concentrations below 1 mM cause 70S ribosome subunit dissociation followed by unfolding. However, magnesium is not the sole component responsible for proper ribosome activity. Early studies demonstrated that polyamines, particularly spermidine or spermine, can compensate for Mg2+ ions for optimum protein synthesis in in vitro translation systems. The highest rate of protein synthesis in vitro, however, is achieved in the presence of Mg2+, polyamines and monovalent cations (K+/NH4 +) together. Similarly, the complete substitution of Mg2+ by polyamines leads to inactivation and loss of integrity of ribosomal subunits in E. coli. Polyamines are known to associate stably and abundantly with ribosomes but are very rarely detected in structural studies and even then ambiguously, hence it was proposed that most polyamine binding sites are differentially occupied in a stochastic manner. The other key players of ribosome activity and stability are monovalent ions. In the absence of K+ ions for example, mammalian ribosomes irreversibly lose their poly-Phe polymerizing activity, while E. coli ribosomes dissociate into subunits upon exposure to very high K+ concentrations or moderate Na+ concentrations. Thus, none of the individual components, cations or polyamines, can entirely substitute for each other, and efficient translation by the ribosome can only be achieved by correct concentrations and balance between them. Despite the vast biochemical data regarding the importance of metal ions for effective ribosome performance and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the identification of metal ions within the ribosome remains elusive due to methodological limitations. Therefore, in the majority of ribosome models derived from conventional data collection used for X-ray structures, metal ions are usually assigned as magnesium—the best-known RNA-stabilizing atom. Consequently, the local chemical environment of the metal ions was interpreted from the point of view of octahedral coordination.


Potassium ions in the peptidyl transferase center. 
a Mapped K+ on secondary structure representation of PTC. 
b Interface view of the 50S (23S, 5S in light blue, proteins in blue) with three tRNAs. In close up view only PTC and K+ ions (with increased van der Waals radius) are shown. 
c Interface view of the 50S, with central protuberance omitted (colors are as in b)

Potassium ions are involved in the stabilization of main functional ligands such as messenger RNA and transfer RNAs, as well as ribosomal RNAs and ribosomal proteins, via the interaction with nitrogen and oxygen atoms of side-chain residues, nucleotide bases, polypeptide or sugar-phosphate backbones. These observations suggest more global and general functions of K+ ions in ribosomal organization rather than its role as a stabilizer of particular regions of the ribosome or particular type of interactions.

https://sci-hub.ee/10.1038/s41467-019-10409-4

Robert Root-BernsteinThe Ribosome as a Missing Link in Prebiotic Evolution III: Over-Representation of tRNA- and rRNA-Like Sequences and Plieofunctionality of Ribosome-Related Molecules Argues for the Evolution of Primitive Genomes from Ribosomal RNA Modules 2019 Jan 2
.]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337102/#:~:text=While%20the%20genes%20encoding%20ribosomal,regions%20%5B41%2C42%5D.

Orthologous genes conserved across all species constitute what is called the Universal Gene Set of Life (UGSL), and consists of less than 100 genes. Not surprisingly, the UGSL is dominated by translation-related genes. These ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and ribosomal proteins make up the most abundant macromolecular species in all living organisms. More surprisingly, as we will demonstrate here, macromolecules based on rRNA-like, tRNA-like and ribosomal-protein-like modules also dominate the rest of the genome, playing roles far removed from translation. We argue that this over-abundance of ribosome-like sequences can only be explained if ribosomes evolved prior to cells providing, through an rRNA-based genome, the basis from which subsequent cellular genomes were elaborated. We call this ribosomal origin of the genome the “ribosome-first” theory of the origins of cellular life.

Gerald F. Joyce and Jack W. Szostak Protocells and RNA Self-Replication 2018
Reconstituting a fully functional and replicating cell from purified biological components is a major challenge and has inspired efforts to simplify aspects of cellular metabolism that seem gratuitous. For example, many chemical modifications of ribosomal RNA (rRNA) are essential for ribosome assembly, but recently Ichihashi and colleagues were able to evolve a variant small subunit rRNA that lacks modifications and can still be efficiently assembled into ribosomes (Murase et al. 2018). If this approach can be extended to the large subunit rRNA, ribosome assembly will be greatly simplified. Similar approaches are aimed at evolving a simpler ribosome, comparable with evolutionary intermediates in the path from the first peptidyl transferase ribozyme to the modern ribosome. Such approaches may contribute to the development of a comprehensive picture of possible paths from simple protocells to complex RNA world cells, and ultimately to the evolution of modern cells that contain DNA genomes, instructed protein synthesis, and a complex metabolism.
https://cshperspectives.cshlp.org/content/10/9/a034801.full#aff-2

Reconstructing the Primordial Ribosome, Tunnel and All
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390850/

The irreducibly complex ribosome is a unique creation in the three domains of life
https://creation.com/ribosomes-and-design

Weizmann Institute of Science: The origin of life in an RNA pocket SEPTEMBER 14, 2022

https://reasonandscience.catsboard.com/t1661p25-translation-through-ribosomes-amazing-nano-machines#9532

https://phys.org/news/2022-09-life-rna-pocket.html

Claim: This story begins several billion years ago. There's only chemistry, no biology—that is, plenty of chemical compounds exist on Earth, but life hasn't yet emerged. Then, among myriads of randomly self-assembled chemical structures, one tiny RNA molecular machine reveals itself as perfectly suitable for creating bonds between activated amino acids, the building blocks of future proteins. It's a turning point in the story of our planet: The synthesis of proteins, biological molecules essential for life, can now begin. Prof. Ada Yonath and her team at the Weizmann Institute of Science believe they have recreated that moment in the lab, showing how it might have happened. As to the primordial peptide-bond-making machine, they discovered that it's still present in virtually every cell of all living organisms, from bacteria to plants and animals, including ourselves. The theory behind the team's project was born some 20 years ago out of startling findings made in Yonath's lab in the course of determining the structure and function of the ribosome—research that would later earn her the 2009 Nobel Prize in Chemistry. The ribosome performs one of life's core processes: manufacturing proteins based on the information in DNA. "We discovered a surprising feature in the structure of this protein-making machine," says Dr. Anat Bashan, a senior staff scientist in Yonath's lab in Weizmann's Chemical and Structural Biology Department. "The ribosome is an enormous macromolecule that is not at all symmetrical; at its heart, it contains two semisymmetrical elements joined together to form a pocket."

Yonath says that they "were particularly struck by the fact that this pocket is found in the ribosomes of all organisms. And this is where all the action takes place—it's where all peptide bonds that create the chain of linked amino acids making up a protein are formed. That's why this basic machinery seems to have persisted unchanged throughout evolution." Meet the protoribosome: That's what the scientists call this pocket-like structure. They hypothesized that the protoribosome is the ancestor of all modern ribosomes, an ancient machine dating back to the prelife period. In comparison to the size of the cell, the protoribosome is rather small. It consists of RNA molecules comprising some 120 nucleotides, about 60 for each of its two semisymmetrical components, which accounts for less than 5 percent of the modern ribosome's dimensions: some 4,500 nucleotides in bacteria and nearly 6,000 in humans

Here's how the scenario unfolded, according to Yonath and her team: The protoribosome came about when a bunch of RNA nucleotide chains self-assembled into two semisymmetrical walls hooked up to create a pocket. Endless other structures must have accidentally self-assembled around the same time, but the protoribosome survived, "going viral," it seems, because it performed useful functions and, thanks to RNA's intrinsic capabilities, it could self-replicate.

Comment:  To get a structure of 120 ribosomal RNAs, is in the realm of the impossible.

The RNA world - a failed hypothesis
https://reasonandscience.catsboard.com/t2024p25-the-rna-world-and-the-origins-of-life#9366

But let us hypothesize that a bunch of RNAs were extant prebiotically, and stable for enough time.

In 1971, Nobel Laureate Manfred Eigen described how the length of a prebiotic information molecule like RNA must fundamentally be limited according to the error rate during replication. Reproduction of longer molecules introduces more errors, and too many errors would lead to an exponential increase in errors over many generations—an error catastrophe. Eigen observed that living organisms require error correction during replication to avoid error catastrophe in long DNA molecules. Yet the error-correction mechanisms themselves must be coded in the same lengthy DNA molecules. This led to Eigen’s paradox: a self-replicating molecule faces a practical size limit of about one hundred nucleotides unless there are error-correction systems, but the error-correction systems themselves must be coded in molecules that are substantially longer than the practical limit. Eigen’s paradox only addresses errors during replication and the need for error-correction mechanisms during replication. The additional burden of accumulated molecular damage from radiation, oxidation, alkylation, chemical mutagens, pathogens, and water, especially over deep time, and the associated requirement for additional molecular-repair mechanisms, greatly compounds Eigen’s paradox.

Eugene V. Koonin: The Logic of Chance: The Nature and Origin of Biological Evolution
The primary incentive behind the theory of self-replicating systems that Manfred Eigen outlined was to develop a simple model explaining the origin of biological information and, hence, of life itself. Eigen’s theory revealed the existence of the fundamental limit on the fidelity of replication (the Eigen threshold): If the product of the error (mutation) rate and the information capacity (genome size) is below the Eigen threshold, there will be stable inheritance and hence evolution; however, if it is above the threshold, the mutational meltdown and extinction become inevitable (Eigen, 1971). The Eigen threshold lies somewhere between 1 and 10 mutations per round of replication (Tejero, et al., 2011); regardless of the exact value, staying above the threshold fidelity is required for sustainable replication and so is a prerequisite for the start of biological evolution

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

Claim: When two activated amino acids happened to interact with one another within this pocket, they formed a bond, facilitated by the prevailing chemical conditions. 
Comment: This is just ad-hoc story telling.

Peptide Bond Formation of amino acids in prebiotic conditions: another unsurmountable problem of protein synthesis on early earth
https://reasonandscience.catsboard.com/t2130-peptide-bonding-of-amino-acids-to-form-proteins-and-its-origins#6664

1. The synthesis of proteins and nucleic acids from small molecule precursors, and the formation of amide bonds without the assistance of enzymes represents one of the most difficult challenges to the model of pre-vital ( chemical) evolution, and for theories of the origin of life.
2. The best one can hope for from such a scenario is a racemic polymer of proteinous and non-proteinous amino acids with no relevance to living systems.
3. Polymerization is a reaction in which water is a product. Thus it will only be favored in the absence of water. The presence of precursors in an ocean of water favors depolymerization of any molecules that might be formed.
4. Even if there were billions of simultaneous trials as the billions of building block molecules interacted in the oceans, or on the thousands of kilometers of shorelines that could provide catalytic surfaces or templates, even if, as is claimed, there was no oxygen in the prebiotic earth, then there would be no protection from UV light, which would destroy and disintegrate prebiotic organic compounds. Secondly, even if there would be a sequence, producing a functional folding protein, by itself, if not inserted in a functional way in the cell, it would absolutely no function. It would just lay around, and then soon disintegrate. Furthermore, in modern cells proteins are tagged and transported on molecular highways to their precise destination, where they are utilized. Obviously, all this was not extant on the early earth.
5. To form a chain, it is necessary to react bifunctional monomers, that is, molecules with two functional groups so they combine with two others. If a unifunctional monomer (with only one functional group) reacts with the end of the chain, the chain can grow no further at this end. If only a small fraction of unifunctional molecules were present, long polymers could not form. But all ‘prebiotic simulation’ experiments produce at least three times more unifunctional molecules than bifunctional molecules.

Claim:  Those amino acids may have arisen on Earth or, as some argue, landed with asteroids from outer space, but their origin is irrelevant to our story. What matters is that within the protoribosome, two activated amino acids could bind to each other. Later on, such bond making united many more amino acids, linking them into a chain.
Comment:  What they don't tell, is how such bonding is precisely orchestrated in modern ribosomes.

Quantum mechanic glimpse into peptide bond formation within the ribosome shed light on origin of life
https://reasonandscience.catsboard.com/t1661-translation-through-ribosomes-amazing-nano-machines#8027


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

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

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

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

Claim:  That was the beginning of proteins, some of which then proved useful in stabilizing and bolstering the protoribosome itself. The proteins kept adding to the protoribosome's structure, enabling it to evolve into the sophisticated protein-making factory it is today. "A modern ribosome can be described as an onion, with the protoribosome at its center, surrounded by more layers that were added during evolution," Yonath says.
Comment: The authors also do mislead by making believe that evolution was a driving agent prior to life starting. Not good.

Claim: In time, protein production would become efficient enough to create chains up to thousands of amino acids in length, and to do so according to the instructions written in the genetic code. 
Comment:  Right. And the genetic code emerged from nowhere?

Claim: The tens of thousands of proteins in the human body all perform different functions, from hormones such as insulin, to antibodies in the immune system, to structural components of cells and tissues such as hemoglobin or the collagen of skin. And they all began with the bonds that once upon a time were formed by chance within the protoribosome.

Fast forward a few billion years. The theory had been upheld by calculations, deductions and dimerization experiments, in which the scientists created RNA dimeric constructs, mimicking the proposed protoribosome. But until recently, the theory's tenets remained, well, largely theoretical. Now, in a new study led by postdoctoral fellow Dr. Tanaya Bose in Yonath's lab, the scientists have provided it with experimental support.

Bose and colleagues set out to explore whether a lab-synthesized protoribosome would be sufficiently stable to survive and perform its presumed bond making, that is, they wanted to find out whether it was indeed capable of this task. She and the other members of Yonath's team created a design for a potential protoribosome by analyzing the pocket-like structures at the heart of several bacterial ribosomes. Bose then prepared these protoribosomal constructs in a laboratory dish.

Comment: 



Next came the crucial step: checking whether the pocket-like dimer constructs could create bonds between amino acids. After mixing the constructs with a solution containing activated amino acid substrates, as well as various salts and other reagents, Bose subjected the products of the reaction to a battery of tests, including mass spectrometry analysis.

To the team's delight, the synthetic protoribosomes created in the lab rose to the task. "Peptide bond formation is the most vital activity in any cell, and we've shown that it can take place within a protoribosome," Bose says.

"The proposed protoribosome might be the missing link between an RNA-dominated world—one that may have existed before proteins and DNA appeared—and life that is based on proteins and nucleic acids, as we know it today," Bashan says.

Comment: The making of pseudo science 1
https://reasonandscience.catsboard.com/t1993-the-make-of-pseudo-science

When certain biologists discuss the early stages of life there is a tendency to think too vaguely. They see a biological wonder before them and they tell a story about how it might have come to be. They may even draw a picture to explain what they mean. Indeed, the story seems plausible enough, until you zoom in to look at the details. I don't mean to demean the intelligence of these biologists. It's just that it appears they haven't considered things as completely as they should. Like a cartoon drawing, the basic idea is portrayed, but there is nothing but blank space where the profound detail of biological processes should be.

Question: What are the different functions of the ribosome ?
ChatGTP: Ribosomes are cellular structures responsible for protein synthesis, and they play a vital role in the translation of genetic information from DNA to proteins. Here are some of the key functions of the ribosome:

Protein synthesis: The primary function of the ribosome is to catalyze the synthesis of proteins from amino acids, using information from messenger RNA (mRNA) molecules. Ribosomes bind to mRNA at the start codon, and they move along the mRNA molecule, matching the sequence of three nucleotides (codons) on the mRNA with corresponding amino acids that are brought to the ribosome by transfer RNA (tRNA) molecules. The ribosome then joins the amino acids together in a specific sequence to form a protein.

Quality control: The ribosome has a quality control mechanism that helps to ensure that only properly folded proteins are released from the ribosome. During the process of protein synthesis, the ribosome checks the folding of the protein as it emerges from the ribosome, and if the protein is not folded correctly, the ribosome can stall and trigger a process that leads to degradation of the protein.

Regulation of gene expression: The rate of protein synthesis by ribosomes can be regulated to control gene expression. For example, the availability of nutrients or other cellular signals can affect the rate of translation by ribosomes, which in turn can affect the levels of specific proteins in the cell.

Antibiotic target: Ribosomes are the target of many antibiotics that are used to treat bacterial infections. Antibiotics can bind to the ribosome and interfere with the process of protein synthesis, thereby inhibiting the growth of bacteria.

In summary, ribosomes are essential structures for protein synthesis in all living organisms, and they perform a variety of important functions in the cell.

Question:How many signalling networks operate in the ribosome?
ChatGTP: The ribosome is a complex molecular machine that plays a crucial role in the translation of genetic information from mRNA to protein. It contains multiple structural and functional components, including ribosomal proteins, rRNA molecules, and various associated factors.

As for signaling networks in the ribosome, there are a few different levels at which signals can be transmitted and received. For example, there are signals related to the initiation and termination of translation, as well as signals related to the quality control of protein synthesis.

However, the specific number of signaling networks that operate in the ribosome is difficult to quantify. This is because the ribosome is a highly dynamic and complex system that interacts with multiple other cellular components and pathways. Additionally, our understanding of the ribosome and its signaling capabilities is still an active area of research, with many open questions and ongoing studies.

Question: How many proteins and cofactors are involved in the synthesis of the ribosome ?
ChatGTP: The synthesis of the ribosome is a complex and highly regulated process that involves numerous proteins and cofactors. The specific number of proteins and cofactors can vary depending on the organism and the type of ribosome being synthesized.

In general, the synthesis of ribosomes involves the coordinated action of a large number of proteins, including chaperones and other factors that facilitate the folding and assembly of ribosomal RNA (rRNA) and ribosomal proteins.

In bacteria, for example, the synthesis of the ribosome involves over 50 proteins and multiple RNA molecules. In eukaryotes, the process is even more complex and involves hundreds of proteins, including many that are involved in the modification and processing of rRNA precursors.

Cofactors such as metal ions and other small molecules may also be involved in ribosome synthesis, although their specific roles can vary. Overall, the synthesis of the ribosome is a complex and highly regulated process that involves many different molecular components.

Question: What is it that does the reading of the codons in the Ribosome?
Answer: The ribosome is responsible for translating the sequence of codons in mRNA into a sequence of amino acids that make up a protein. During translation, the ribosome recognizes each codon by interacting with the corresponding anticodon on a transfer RNA (tRNA) molecule.

tRNA molecules are small RNA molecules that carry a specific amino acid and have an anticodon that base pairs with the codon on the mRNA. Each tRNA molecule is specific for one type of amino acid and has a unique anticodon that corresponds to a specific codon on the mRNA.

When a ribosome encounters a codon on the mRNA, it recruits a complementary tRNA molecule with the appropriate anticodon and attached amino acid. The ribosome then forms a peptide bond between the amino acid on the incoming tRNA and the growing peptide chain on the previous tRNA molecule, releasing the previous tRNA molecule and continuing along the mRNA to the next codon. This process repeats until the ribosome reaches a stop codon, at which point the protein is released.

Question: How does that recognition occur?
Answer: The recognition between the codon and anticodon occurs through complementary base pairing between the nitrogenous bases of the mRNA codon and the tRNA anticodon. The ribosome has two subunits, each with specific binding sites for the mRNA and tRNA molecules. The A site (aminoacyl site) binds to the incoming aminoacyl-tRNA, which carries the appropriate amino acid for the current codon. The P site (peptidyl site) binds to the tRNA molecule that is carrying the growing polypeptide chain.

The recognition of the codon and anticodon involves specific interactions between the bases of the mRNA codon and the tRNA anticodon. For example, if the mRNA codon is AUG (which codes for the amino acid methionine), the corresponding tRNA molecule would have the anticodon UAC. The ribosome recognizes the AUG codon and recruits the UAC anticodon, allowing the incoming tRNA molecule to bind to the A site of the ribosome.

Once the incoming tRNA molecule is bound to the A site, the ribosome catalyzes the formation of a peptide bond between the amino acid carried by the incoming tRNA and the growing polypeptide chain on the tRNA molecule in the P site. This process transfers the amino acid from the tRNA molecule in the A site to the growing polypeptide chain, and the ribosome moves along the mRNA to the next codon.

This process of codon-anticodon recognition and peptide bond formation continues until a stop codon is reached, at which point the ribosome releases the completed polypeptide chain.

Question: What mechanism does the recognition of the codon , in order to recruit the corresponding tRNA ?
Answer: The recognition of the codon by the ribosome occurs through a combination of base-pairing interactions and conformational changes in the ribosome itself.

When a codon in the mRNA enters the ribosome's decoding center, it interacts with the anticodon loop of a charged tRNA that is complementary to the codon. The recognition process involves several steps:

The ribosome's small subunit binds to the mRNA molecule and scans along it until it reaches the start codon (AUG) that signals the beginning of the coding sequence.
When the ribosome encounters the start codon, it recruits a charged initiator tRNA molecule that recognizes the AUG codon. This tRNA molecule carries the amino acid methionine, which is the first amino acid in most proteins.
The initiator tRNA molecule base-pairs with the AUG codon on the mRNA, positioning the methionine residue in the ribosome's P-site.
The ribosome's large subunit then joins the complex, forming a complete ribosome with the initiator tRNA molecule in the P-site and the A-site available to accept the next tRNA molecule.
As the ribosome moves along the mRNA molecule, it reads each codon and matches it with the corresponding anticodon on a charged tRNA molecule that carries the appropriate amino acid.
The ribosome ensures the correct match by using proofreading mechanisms, which check the base-pairing between the codon and anticodon, as well as the amino acid attached to the tRNA molecule.

Error check and repair during protein synthesis


1. Pre-translation Quality Control

These mechanisms ensure that all components needed for translation are properly prepared before actual protein synthesis begins.

Prokaryotic-Exclusive Mechanisms

Ribosomal Subunit Assembly Checkpoints
Small Subunit (30S) Assembly Checkpoints: Assembly factors and chaperones aid in the correct assembly of the 30S subunit.

The assembly of the 30S ribosomal subunit in prokaryotes involves a series of complex and interdependent events. To ensure that the 30S subunit assembles correctly, there are quality control mechanisms or "checkpoints" in place. Some key players involved in these assembly checkpoints include:

RNA helicases: These proteins can modify the structure of rRNA to ensure it's correctly folded. An example in E. coli is DeaD/CsdA.
16S rRNA methyltransferases: Methylation can serve as a checkpoint for the proper folding or assembly of rRNA. RsmA/KsgA is an example that methylates two adjacent adenosines (A1518 and A1519) in 16S rRNA. If the 30S subunit doesn't pass this checkpoint, the ribosome becomes kasugamycin-sensitive.
GTPases: For instance, RsgA/YjeQ interacts with the 30S subunit. Mutations affecting its function result in 30S particles missing multiple ribosomal proteins, suggesting it might serve as a checkpoint for the proper assembly of certain proteins onto the subunit.
Endonucleases: RNase R is an example. It's involved in the degradation of improperly formed 16S rRNA precursors.

These proteins and enzymes serve to monitor and ensure the fidelity of the 30S subunit assembly. They can either promote the correct assembly, inhibit the incorporation of faulty components, or target incorrectly assembled particles for degradation.

Large Subunit (50S) Assembly Checkpoints: Chaperones and assembly factors ensure the correct assembly of the 50S subunit.

The assembly of the large subunit (50S) of the prokaryotic ribosome is even more intricate than the small subunit. There are several proteins and assembly factors that are specifically involved in error-checking and facilitating the correct assembly of the 50S subunit in prokaryotes:

Era GTPase: A well-conserved GTPase that is vital for the maturation of the 50S subunit in E. coli.
Ribosomal RNA methyltransferases: Proteins like RlmN and RlmO play roles in methylation of the 23S rRNA, a component of the 50S subunit.
Riboflavin synthase: RimP is an important factor required for the late-stage assembly of the 50S ribosomal subunit. In the absence of RimP, a few ribosomal proteins fail to assemble into the 50S subunit.
Peptide chain release factors: PrmC (also known as HemK) modifies peptide chain release factors which interact with the 50S subunit.
RbgA (RrbA in E. coli): A universally conserved GTPase, which is essential for the assembly of the 50S subunit, especially in bacteria. In its absence, the assembly process is stalled.
HelD: An RNA helicase involved in the maturation of the 50S subunit.
Endonucleases like RNase III: They participate in the maturation of the 23S and 5S rRNAs.
L16: A ribosomal protein that acts as a checkpoint by ensuring the proper incorporation of other ribosomal proteins.
Assembly GTPases: Such as EngA, EngB (Der), and ObgE, which play a role in 50S subunit biogenesis.

These molecules collectively ensure that the 50S subunit assembles properly. When errors are detected, many of these proteins either actively correct the issue, inhibit the incorporation of incorrectly folded or modified components, or guide the misassembled particles toward degradation or recycling.

70S Ribosome Assembly Quality Control: Checkpoints ensure that correctly assembled subunits form the functional 70S ribosome.

The quality control during the 70S ribosome assembly is not as extensively studied as the individual 30S and 50S subunits, but there are still known players that ensure proper assembly and function of the 70S ribosome:

IF3 (Initiation Factor 3): This is a pivotal factor for the 30S subunit. It prevents premature association of the 30S and 50S subunits and ensures that only properly formed 30S subunits (which have the correct tRNA and mRNA in place) can combine with the 50S subunits to form an active 70S initiation complex.
RsfS (or YbeB in E. coli): This protein is believed to act as a negative regulator of 70S ribosome assembly. RsfS binds to the 30S subunits, preventing them from associating with the 50S subunits, ensuring that only mature 30S subunits are involved in 70S ribosome formation.
RimM: Primarily known for its role in 30S assembly, it also aids in efficient 70S ribosome assembly by ensuring the proper alignment and maturation of the 30S subunit.
RbfA (Ribosome-binding factor A): This is another protein that is known to associate with the 30S subunit but also plays a role in ensuring proper 30S and 50S subunit association.
ERA GTPase: Along with its role in 30S and 50S maturation, Era also ensures the correct assembly of the 70S ribosome. It binds to 16S rRNA and the 30S subunit, ensuring proper subunit interaction.
The GTPases - EngA, Der, ObgE, and RbgA: These have been mentioned in the context of individual subunit assembly, but they also likely play a role in ensuring the proper assembly and function of the 70S ribosome.

The above players work in tandem to ensure that only properly assembled 30S and 50S subunits come together to form the functionally active 70S ribosome. If there are errors in either of the subunits or their assembly, these factors inhibit the formation of the 70S ribosome, thus acting as checkpoints.

Ribosome Subunit Association Control
Anti-Association Factors in Ribosome Assembly: Proteins in prokaryotic cells that prevent premature association of ribosomal subunits.

The ribosome subunit association control in prokaryotic cells involves factors that prevent the premature association of the 30S and 50S subunits until they are properly assembled and ready for translation. Here are the known anti-association factors:

IF3 (Initiation Factor 3): This is the primary and most well-studied anti-association factor. IF3 binds to the 30S subunit and prevents its premature association with the 50S subunit. It ensures that only properly formed 30S subunits (with the correct tRNA and mRNA in place) can combine with the 50S subunits to initiate translation.
RsfS (or YbeB in E. coli): This protein acts as a negative regulator of ribosome assembly. RsfS binds to the 30S subunits, preventing them from associating with the 50S subunits, ensuring that only mature 30S subunits are involved in ribosome formation.

These are the major players that work as checkpoints to ensure that premature ribosomal subunit association does not occur in prokaryotic cells. Their action is crucial for maintaining the fidelity and efficiency of protein synthesis.

mRNA and tRNA Interaction with the Ribosome

Anti-Shine-Dalgarno Sequence in tRNAs: In bacteria, this sequence in tRNAs prevents them from binding to the ribosome mRNA binding site.

The Shine-Dalgarno (SD) sequence in bacterial mRNAs plays a key role in initiating translation by pairing with a complementary anti-Shine-Dalgarno (aSD) sequence located at the 3' end of the 16S rRNA of the 30S ribosomal subunit. This interaction helps to correctly position the start codon at the ribosome's P-site, allowing for the correct initiation of protein synthesis.

However, to prevent spurious binding of tRNAs to the mRNA binding site of the ribosome (which would disrupt the initiation of translation), many bacterial tRNAs possess an anti-Shine-Dalgarno (aSD)-like sequence. This sequence competes with the mRNA's SD sequence for binding to the ribosome's aSD sequence, thereby preventing incorrect initiation events.

In terms of "error-checking" or players involved:

16S rRNA: Contains the anti-Shine-Dalgarno sequence that pairs with the SD sequence in mRNAs to ensure correct positioning during the initiation of translation.
tRNAs with aSD-like sequences: These tRNAs can bind to the aSD site on the ribosome, preventing other interactions and thereby ensuring the fidelity of translation initiation.
The system works not so much by "error-checking" but by competition and ensuring that the right interactions occur at the right time to initiate translation correctly. The presence of aSD-like sequences in tRNAs provides an additional layer of regulation to ensure that mRNA is accurately and efficiently translated.

tRNA Charging and Accuracy
tRNA Charging Fidelity: Mechanisms in prokaryotic cells that ensure the correct amino acid is attached to its corresponding tRNA.

The process of attaching the correct amino acid to its corresponding tRNA molecule is termed "tRNA charging" or "aminoacylation". This process is critical to ensure the fidelity of protein synthesis. 

Aminoacyl-tRNA synthetases (aaRSs): These are the primary enzymes responsible for tRNA charging. Each type of aminoacyl-tRNA synthetase recognizes a specific amino acid and its corresponding tRNA(s). It ensures that the correct amino acid is attached to the 3' end of the correct tRNA.
Editing Sites of aaRSs: Some aminoacyl-tRNA synthetases have editing sites separate from their synthetic active sites. If a wrong amino acid is initially attached to a tRNA, these editing sites can remove it before the tRNA leaves the enzyme, ensuring an additional level of accuracy.
ATP: It provides the energy for the reaction. Amino acids are first activated by ATP to form aminoacyl-AMP, which is then transferred to the tRNA.
tRNA elements: Specific anticodons and acceptor stem sequences in tRNAs are recognized by the aaRSs. These elements ensure that each tRNA interacts with its cognate aaRS.
Post-transcriptional modifications: Some tRNA nucleotides undergo modifications after transcription. These modifications can affect the recognition by aaRSs and contribute to the accuracy of tRNA charging.

Together, these components and mechanisms ensure a high level of fidelity in the tRNA charging process in prokaryotic cells, making sure that the right amino acid is attached to its corresponding tRNA, which is crucial for the accuracy of protein synthesis.

Pre-translation Quality Control in Eukaryotes

These mechanisms ensure all components needed for translation are correctly prepared and functional before actual protein synthesis begins.

Ribosome biogenesis: Quality control mechanisms survey nascent ribosomes for their functionality. 7

Surveillance Factors

U3 snoRNP: Involved in early steps of 18S rRNA processing. A defect here would lead to a discard of the pre-rRNA.
Nop53p: Serves as a checkpoint protein for the proper modification and processing of the 5.8S and 25S rRNAs.

Endonucleolytic Cleavage

RNase MRP: Targets pre-rRNA for cleavage in the ITS1 region.
Las1: Involved in cleavage at the C2 site in ITS2.

Exosome Complex

Targets aberrant RNA molecules for degradation. In ribosome biogenesis, the exosome is critical for the correct processing of the 3' end of 5.8S rRNA. Components include RRP44/Dis3, RRP6, and core exosome subunits.
Dom34-Hbs1: Targets stalled 80S ribosomes, facilitating ribosome recycling. Though this complex mainly functions in translation, it serves as a backup system for ribosomes that have bypassed other quality control mechanisms and reached the translation initiation phase.
SSU Processome: A massive ribonucleoprotein complex responsible for the biogenesis of the small 18S rRNA subunit. Any defects in this process will result in nonfunctional rRNA and its subsequent discard.

Nucleolar Surveillance

Rrp5: Tied to the initial cleavage steps of pre-rRNA. If these steps are compromised, further downstream processing of rRNA is blocked.
Nsa2: Functions as a sensor for the 27S pre-rRNA. In its absence, these pre-rRNAs are subject to degradation.
Exportin (Xpo1/Crm1): Responsible for the nuclear export of ribosomal subunits. Defects in subunit biogenesis can lead to export blocks.

GTPases

Nog1: A GTP-binding protein, critical for large subunit biogenesis. If this protein is mutated, it causes accumulation of pre-rRNA intermediates which are then targeted for degradation.

ESCs (Eukaryotic-Specific Elements)

These are eukaryotic-specific expansion segments and are subject to tight quality control. Any defects can result in nonfunctional rRNA.
Ltv1, Rrp12, and Tsr1: These are checkpoint proteins specifically for the small subunit. Their absence or mutation results in a halt in the 40S subunit's maturation, leading to degradation of pre-40S particles.
The above-mentioned factors ensure that only correctly processed and assembled ribosomal subunits become functional in the cytoplasm. Any aberrant ribosomal particles, whether due to defective rRNA or assembly factors, are promptly targeted for degradation, ensuring the high fidelity required for effective and accurate protein synthesis.

Monitoring of Ribosome Biogenesis in the Nucleolus

The nucleolus, where ribosomes are synthesized, ensures correct ribosome biogenesis. Ribosome biogenesis in the nucleolus is a multi-faceted process that involves numerous checkpoints and quality control mechanisms. Let's hone in on the specific players involved in error-checking, quality control, repair, and discard/recycling during ribosome biogenesis in the nucleolus, which is a part of pre-translation quality control in eukaryotes:

Surveillance and Discard Mechanisms

Exosome Complex: A multi-protein complex that degrades aberrant rRNA molecules. It includes subunits like RRP44/Dis3 and RRP6.
Mtr4: An RNA helicase that cooperates with the exosome to target defective rRNAs for degradation.

Quality Control during Pre-rRNA Processing

U3 snoRNP: Ensures the correct early processing of 18S rRNA. Aberrations here trigger pre-rRNA decay.
Rrp5: Interacts with U3 snoRNP and monitors the initial cleavage steps of pre-rRNA. Defective processing will halt further maturation.

Nucleolar Retention

Aberrant ribosomal subunits or pre-rRNAs are retained in the nucleolus, preventing their export to the cytoplasm. This retention serves as a quality control measure.

Nucleophosmin (B23): A multifunctional nucleolar protein that can bind immature ribosomal subunits, potentially preventing their premature export.

Checkpoints for Large Subunit (60S) Biogenesis

Nog1: A GTPase that is essential for 60S ribosomal subunit biogenesis. Aberrations in its function cause pre-rRNA accumulation and degradation.
Nsa2: Monitors the maturation of the 27S pre-rRNA.


Checkpoints for Small Subunit (40S) Biogenesis

Tsr1 and Ltv1: Both are critical during the late stages of 40S subunit maturation. Defects or absence of these proteins lead to degradation of the 20S pre-rRNA.
Rio2: A kinase that checks the proper maturation of the 20S pre-rRNA.

rRNA Modification Checkpoints

Small Nucleolar RNPs (snoRNPs): These are involved in site-specific modifications of rRNAs, including methylation and pseudouridylation. Proper modification is crucial for rRNA maturation and function.

Repair Mechanisms

While direct repair mechanisms for rRNA are not well-defined, improper modifications or errors can lead to degradation pathways being initiated, thereby ensuring only intact and properly modified rRNAs are incorporated into ribosomes.

Ribosome Assembly Factors

Numerous assembly factors temporarily associate with pre-ribosomal particles to ensure proper maturation and assembly. If ribosomal proteins or rRNAs are misfolded or improperly assembled, these assembly factors can retain the defective subunits in the nucleolus for degradation. The nucleolus has an intricate network of these and other factors that meticulously ensure every step of ribosome biogenesis is error-free. Given the essential role of ribosomes in protein synthesis, the eukaryotic cell invests considerable resources to guarantee that only fully functional ribosomes are produced. Any errors in this process could lead to larger downstream issues, such as mistranslation or cellular stress.

Regulation of Ribosome Biogenesis by Non-coding RNAs

Non-coding RNAs influence the synthesis and assembly of ribosomes. Non-coding RNAs (ncRNAs) have been recognized as significant players in cellular processes, including the regulation of ribosome biogenesis, especially within the nucleolus. They play roles in facilitating the correct processing, modification, and assembly of ribosomal RNA (rRNA) and ribosomal subunits, as well as in quality control mechanisms. Here are the key non-coding RNAs and associated players related to the monitoring of ribosome biogenesis in the nucleolus:

Small Nucleolar RNAs (snoRNAs): Involved in the site-specific modification (methylation and pseudouridylation) of rRNA molecules. Defects in snoRNA-guided modifications can lead to rRNA degradation to eliminate improperly modified rRNAs. They often associate with specific proteins to form snoRNP complexes. Common proteins include fibrillarin (for methylation) and dyskerin (for pseudouridylation).
External and Internal Transcribed Spacers (ETS and ITS): These are transcribed as part of the primary rRNA transcript but are later removed. They guide the correct folding and processing of the rRNA. Misprocessed or misfolded pre-rRNAs due to issues with spacers are targeted for degradation.
U3 snoRNA: Plays a crucial role in the early cleavage events of pre-rRNA processing, ensuring proper 18S rRNA formation. Malfunctions in U3 snoRNA interaction with pre-rRNA can trigger degradation pathways to eliminate faulty rRNAs.
Pescadillo Homolog (PES1): An essential protein associated with snoRNAs that influences rRNA processing.  Mutations or defects in PES1 can disrupt rRNA maturation, leading to nucleolar stress.
5' ETS ncRNAs: These are cleaved segments of the external transcribed spacer regions and play roles in facilitating the proper processing of pre-rRNAs. Aberrant 5' ETS ncRNAs or defects in their function can trigger degradation of associated rRNAs.
RNA Exosome Complex: Not an ncRNA itself, but a multi-protein complex that plays a significant role in degrading aberrant rRNA molecules. It's involved in processing and turnover of various ncRNAs. Components like RRP44/Dis3 and RRP6 play roles in degradation, ensuring quality control by eliminating improperly processed or modified rRNAs.
piRNAs and siRNAsThough their primary roles are in post-transcriptional silencing and protection against transposable elements, emerging evidence suggests they may also play roles in ribosomal quality control, particularly in ensuring the stability of rDNA loci.

In the context of ribosome biogenesis in the nucleolus, these ncRNAs and associated protein factors contribute to a multi-layered quality control system. They ensure that only properly processed, modified, and assembled rRNAs are incorporated into ribosomal subunits, maintaining the integrity of protein synthesis in eukaryotic cells.

rRNA Synthesis Error Check

Surveillance mechanisms ensure only correctly processed and modified rRNA is incorporated into ribosomes. Ribosomal RNA (rRNA) synthesis, processing, and modifications are essential steps in ribosome biogenesis. Eukaryotic cells have established multiple surveillance mechanisms to ensure that only properly processed and modified rRNA is incorporated into ribosomes. Here are some of the key players and mechanisms involved in rRNA synthesis error check:

RNA Exosome Complex: This multi-protein complex is involved in the degradation of aberrant rRNA molecules. Key components include RRP44/Dis3 and RRP6, which play roles in the degradation of improperly processed or modified rRNAs.
Nucleolar Surveillance: Any pre-rRNA that fails to be properly processed is retained in the nucleolus and subsequently degraded. Nucleolar proteins such as NOB1 and XRN2 are involved in the degradation of these aberrant rRNA species.
5' ETS and ITS Checkpoints: The External and Internal Transcribed Spacers (ETS and ITS) guide the correct folding and processing of rRNA. Misprocessed or misfolded pre-rRNAs due to issues with these spacers are targeted for degradation.
U3 snoRNA: Facilitates early cleavage events of pre-rRNA processing. Any misfunctions in U3 snoRNA interaction with pre-rRNA can trigger degradation pathways.
Small Nucleolar RNPs (snoRNPs): Responsible for the site-specific modification of rRNAs. Mis-modified rRNA, due to issues with snoRNPs, can be targeted for degradation. snoRNAs like C/D box and H/ACA box snoRNAs guide these modifications, and associated proteins like fibrillarin and dyskerin facilitate them.
TRAMP Complex: Assists the exosome in targeting aberrant RNAs for degradation. Components include a poly(A) polymerase, a zinc-knuckle protein, and the helicase Mtr4.
UTP-A, UTP-B, and UTP-C Complexes: Involved in early steps of 18S rRNA processing. If these complexes malfunction or if the initial processing steps are hindered, rRNA precursors are retained and degraded.
Nop53p: Acts as a retention factor for improperly processed 60S rRNA precursors, preventing their export from the nucleolus.
Rrp5: Binds to pre-rRNA and assists in its early processing steps. Defects in these steps due to Rrp5 malfunctions can result in rRNA degradation.

The aforementioned mechanisms and players contribute to the rigorous surveillance of rRNA synthesis and processing in eukaryotic cells. This quality control ensures that ribosomes, which are central to the protein synthesis machinery, function efficiently and accurately.

rRNA Secondary Structure Verification

Ensuring rRNA forms the correct secondary structures vital for ribosome function. Ensuring that the rRNA adopts the appropriate secondary structures is essential for the proper functioning of ribosomes. The secondary structure of rRNA dictates its interactions with ribosomal proteins and its ability to carry out its catalytic role in translation. Several factors and mechanisms ensure that these structures are correctly formed:

K-turns and K-loops: Specialized secondary structures in rRNA that allow for specific protein interactions. These motifs are stabilized by proteins like L7Ae in eukaryotes.
Pseudouridines: Pseudouridylation of rRNA plays a significant role in stabilizing certain secondary structures, facilitated by specific snoRNPs.
Base Modifications: Various base modifications, such as methylation, can stabilize or alter rRNA secondary structures. The exact modification is often directed by specific snoRNAs.
Box C/D and Box H/ACA snoRNAs: These snoRNAs guide site-specific modifications in rRNA that can influence its secondary structure. They guide enzymes to specific sites in the rRNA to methylate or pseudouridylate it.
Ribosome Biogenesis Factors: Several proteins involved in ribosome biogenesis, such as Nop58, Nop56, and fibrillarin, bind to rRNA and help it fold into the correct secondary structures.
RNA helicases: These enzymes, like Dbp4 and Mtr4, can remodel rRNA structures, ensuring the proper formation of secondary structures necessary for function.
Endo- and Exonucleases: These enzymes trim the ends of rRNA precursors, which can be essential for the final folding and structure of the mature rRNA. Examples include RNase MRP and Xrn1.
RNA Chaperones: These proteins can bind to rRNA and aid in its folding, ensuring that the correct secondary structures are formed. This category includes proteins like Nhp2 and Snu13.

Ensuring proper rRNA secondary structure is a key component of ribosome quality control. Any disruption in these structures can lead to nonfunctional ribosomes or decreased translation fidelity. The intricate system of modifications, protein interactions, and enzymatic remodeling ensures that rRNA adopts the structures vital for its role in translation.

Modification Checks for rRNA 

Systems ensure necessary modifications to rRNA before incorporation into ribosomal subunits. The proper modification of ribosomal RNA (rRNA) is critical to ensure the efficient and accurate functioning of ribosomes in protein synthesis. Eukaryotic cells have established several mechanisms to verify that rRNA undergoes the necessary modifications prior to its incorporation into ribosomal subunits. Here are the key players and mechanisms involved in this process:

Small Nucleolar RNPs (snoRNPs): These complexes are responsible for the majority of site-specific modifications of rRNAs, particularly methylation and pseudouridylation. There are two major types:

Box C/D snoRNPs: Direct the methylation of specific ribose residues in rRNA. Fibrillarin is a core protein component that serves as the methyltransferase.
Box H/ACA snoRNPs: Guide the isomerization of uridines into pseudouridines in rRNA. The catalytic protein dyskerin carries out the modification.

RNA helicases: Proteins like Dhr1 and Mtr4 are involved in rRNA remodeling. These enzymes ensure that rRNA is properly structured to undergo necessary modifications.
Nop1: A conserved protein associated with Box C/D snoRNPs, playing a role in 2'-O-methylation of ribose sugars in rRNA.
Gar1: Associated with Box H/ACA snoRNPs, it's involved in pseudouridylation of rRNA.
Nucleolar Surveillance: Any rRNA that fails to receive proper modifications is retained in the nucleolus and targeted for degradation. Proteins like XRN2 play a role in the degradation process.
NOP58 and NOP56: Both proteins are core components of Box C/D snoRNPs, and they are involved in rRNA methylation.
TRAMP Complex: Alongside the RNA exosome complex, the TRAMP complex aids in recognizing and degrading improperly modified rRNA.
RNA Chaperones: Proteins that assist rRNA to adopt conformations amenable to modifications, such as Nhp2 and Snu13.
UTP-A, UTP-B, and UTP-C Complexes: These complexes are involved in early stages of rRNA processing. They ensure proper rRNA folding which indirectly ensures that the correct regions of rRNA are accessible for modifications.

The precise modifications of rRNA play crucial roles, from ensuring proper rRNA folding to fine-tuning the ribosome's functional activities during translation. The detailed quality control mechanisms ensure that only appropriately modified rRNA is incorporated into the final ribosomal units, safeguarding the fidelity of protein synthesis.

Ribosomal RNA Integrity Monitoring 

Mechanisms to detect and manage damaged or misfolded rRNA. The integrity of ribosomal RNA (rRNA) is paramount for the proper function of ribosomes in protein synthesis. To ensure that only the correctly folded and undamaged rRNA molecules are incorporated into the ribosomal subunits, eukaryotic cells have implemented several surveillance and repair mechanisms. Here's a breakdown of the key players and systems involved in rRNA integrity monitoring:

RNA Exosome Complex: This multi-protein complex is pivotal for the degradation of damaged or misfolded rRNA molecules. The main components, RRP44/Dis3 and RRP6, are responsible for the degradation of these faulty rRNAs.
Nucleolar Surveillance: Misfolded or damaged rRNA retained in the nucleolus is identified and subjected to degradation. Nucleolar proteins like NOB1 and XRN2 play vital roles in the degradation process.
p53 Protein: Known primarily as a tumor suppressor, p53 also plays a role in the response to ribosomal stress. When rRNA damage or misfolding is detected, p53 can be activated to halt cell cycle progression and allow for repair or removal of the defective rRNA.
Small Nucleolar RNPs (snoRNPs): While primarily responsible for rRNA modification, snoRNPs also play roles in rRNA folding. Misfolded rRNAs might result from improper snoRNP activity, triggering degradation pathways.
Helicases: Enzymes like Dhr1 and Mtr4 not only help in rRNA remodeling but also assist in recognizing and resolving misfolded rRNA structures.
TRAMP Complex: Acting in tandem with the RNA exosome complex, the TRAMP complex aids in recognizing and targeting improperly folded or damaged rRNA for degradation.
RNase MRP: An RNA-protein complex involved in rRNA processing. It ensures the proper maturation of rRNA, and any errors in its function could result in misfolded rRNA.
Nop53p: This protein serves as a retention factor for improperly processed rRNA, ensuring they are kept in the nucleolus and subjected to corrective measures or degradation.
TOR Signaling Pathway: The Target of Rapamycin (TOR) pathway, which governs cellular growth in response to nutrient availability, can be affected by ribosomal stress. Misfolded or damaged rRNA can signal through this pathway to adjust ribosome biogenesis and protein synthesis rates.
The Heat Shock Response: In the event of stress conditions that can damage rRNA, the heat shock response is activated. Molecular chaperones, such as Hsp70 and Hsp90, are upregulated to assist in rRNA refolding and protection.

Ensuring the integrity of rRNA is essential given the central role of ribosomes in protein synthesis. Eukaryotic cells invest significant resources in monitoring, repair, and quality control mechanisms to guarantee that ribosomes operate with the highest fidelity and efficiency.

Monitoring Ribosomal Protein Binding

Ensuring correct ribosomal proteins bind at appropriate sites on rRNA. The correct binding of ribosomal proteins to rRNA is essential for ribosome assembly and functionality. Eukaryotic cells deploy specific mechanisms to guarantee that ribosomal proteins associate correctly with rRNA. Here's an outline of the crucial players and systems involved in monitoring ribosomal protein binding:

Assembly Chaperones: These proteins assist ribosomal proteins in binding to the correct rRNA sites. Examples include Rrp5, which interacts with multiple ribosomal proteins and rRNA to ensure proper assembly.
Nucleolar Surveillance: This mechanism detects any improperly bound ribosomal proteins in the nucleolus. Misassembled particles are retained in the nucleolus, ensuring they don't participate in translation.
Nucleoplasmic Quality Control: If improperly assembled ribosomal subunits escape the nucleolar checkpoints, they can be identified and targeted for degradation in the nucleoplasm before they reach the cytoplasm.
AAA-ATPases: Proteins like Rix7 play a role in remodeling ribosomal subunits and ensuring proper protein binding. They use the energy from ATP hydrolysis to adjust the ribosomal proteins' positions on rRNA.
RACK1: The Receptor for Activated C Kinase 1 (RACK1) is a ribosomal protein that also serves as a scaffold for various signaling proteins. It plays a pivotal role in ensuring that specific ribosomal proteins are appropriately integrated during ribosome assembly.
RNA Exosome Complex: In instances where ribosomal proteins are not correctly bound, the associated rRNA can be subjected to degradation by this complex, ensuring that faulty assemblies do not contribute to functional ribosomes.
Exportin-1 (Xpo1): This is a nuclear export receptor that recognizes properly assembled ribosomal subunits. It ensures that only correctly assembled ribosomal subunits are transported from the nucleus to the cytoplasm.
Nin1: Acts as a ribosome assembly factor and ensures the correct incorporation of ribosomal proteins during the assembly process.
Rrp12: This protein is critical for the correct binding of ribosomal proteins during early assembly stages. Defects in Rrp12 function could result in improper ribosomal protein incorporation.
The TOR Pathway: The Target of Rapamycin (TOR) signaling pathway can be influenced by ribosomal protein binding errors. If there are issues with ribosomal protein assembly, this pathway can modulate ribosome biogenesis accordingly.

Ensuring the precise binding of ribosomal proteins to rRNA is pivotal for the structural and functional integrity of ribosomes. The mentioned mechanisms and players form a robust network of quality control in eukaryotic cells, ensuring that the protein synthesis machinery operates efficiently and accurately.

Nuclear Import of Ribosomal Proteins

Mechanisms ensure ribosomal proteins synthesized in the cytoplasm are imported into the nucleus and nucleolus. Ribosomal proteins are synthesized in the cytoplasm and need to be accurately and efficiently transported to the nucleus and further to the nucleolus for assembly with ribosomal RNA (rRNA). Eukaryotic cells have established specialized mechanisms to regulate the nuclear import of ribosomal proteins. Here are the critical players and systems involved in this process:

Importin-β Family: This family of proteins, particularly Importin-β and Importin-α, plays a central role in transporting ribosomal proteins into the nucleus. Importin-α recognizes the nuclear localization signals (NLS) on ribosomal proteins, while Importin-β mediates transport through the nuclear pore complex.
Nuclear Pore Complex (NPC): A massive protein complex that spans the nuclear envelope, acting as a gatekeeper for the transport of molecules between the nucleus and cytoplasm. Ribosomal proteins pass through NPCs to enter the nucleus.
Ran GTPase Cycle: The Ran GTPase system provides the energy and directionality for nuclear import. In the cytoplasm, Ran is predominantly GDP-bound, while in the nucleus, it is GTP-bound. This gradient is maintained by two key proteins, RanGEF (guanine nucleotide exchange factor) in the nucleus and RanGAP (GTPase-activating protein) in the cytoplasm. The gradient is essential for the release of ribosomal proteins into the nucleus and the recycling of importins back to the cytoplasm.
Nucleophosmin (NPM1/B23): This nucleolar protein plays a role in the shuttling of ribosomal proteins from the nucleoplasm to the nucleolus, ensuring that they reach their final destination for ribosome assembly.
Ribosomal Protein Adaptation: Some ribosomal proteins can be modified, either by phosphorylation or by binding to other proteins, to mask their NLS, regulating their nuclear import.
Nucleolar Importins: Certain importins, such as Importin-7, specialize in the transport of proteins directly into the nucleolus.
Nucleocytoplasmic Shuttling: Some ribosomal proteins can shuttle between the nucleus and cytoplasm, with specific exportins mediating their exit from the nucleus when required.
Ribosome Biogenesis Factors: Numerous proteins, such as Rrp12 and Nmd3, play roles in ensuring that ribosomal proteins are correctly incorporated into maturing ribosomal subunits within the nucleolus.
Feedback Mechanisms: If ribosomal proteins cannot be incorporated into ribosomes, they can accumulate in the nucleoplasm and bind to Mdm2, inhibiting its function. This leads to the stabilization of p53, a transcription factor that can halt cell cycle progression or initiate apoptosis.

Ensuring the precise import of ribosomal proteins from the cytoplasm to the nucleus and nucleolus is a fundamental aspect of cell biology. The mechanisms and players mentioned above facilitate this intricate process, ensuring the fidelity of ribosome biogenesis in eukaryotic cells.

Nuclear Export of Ribosomal Subunits

Only mature ribosomal subunits are exported from the nucleus to the cytoplasm. The export of mature ribosomal subunits from the nucleus to the cytoplasm is a tightly regulated process, ensuring that only fully assembled and functional ribosomes participate in protein synthesis. Here are the key players and mechanisms involved in the nuclear export of ribosomal subunits in eukaryotic cells:

Exportin 1 (CRM1): A major export receptor, Exportin 1, in conjunction with RanGTP, binds to specific nucleoporins and ribosomal subunit export adaptors to facilitate the transport of ribosomal subunits through the nuclear pore complex.
Nuclear Pore Complex (NPC): This massive protein assembly serves as the main gateway for ribosomal subunits to traverse the nuclear envelope. The NPC allows the selective passage of large ribonucleoprotein complexes, like ribosomal subunits, between the nucleus and cytoplasm.
Nmd3: A dedicated adapter protein, Nmd3 binds to the 60S ribosomal subunit and serves as an adaptor for Exportin 1-mediated export.
Arx1: Another adaptor for the 60S subunit, Arx1 acts as an export chaperone that also binds to the ribosomal subunit and assists in its export.
Ran GTPase Cycle: The gradient of RanGTP in the nucleus and RanGDP in the cytoplasm is vital for the release of ribosomal subunits into the cytoplasm and the recycling of exportins back into the nucleus.
Tsr1 and Rrp12: These proteins are involved in the export of the 40S ribosomal subunit. Tsr1 aids in the late stages of 40S maturation, while Rrp12 is crucial for its export.
Mex67-Mtr2: In yeast, this heterodimer acts as an alternative export receptor for the 60S ribosomal subunit. It recognizes specific nucleoporins to facilitate subunit passage through the NPC.
Rrp12: Apart from its role in 40S subunit export, Rrp12 is also involved in the nuclear export of the 60S subunit, ensuring its proper transport to the cytoplasm.
Nucleocytoplasmic Feedback: A feedback mechanism exists where defects in ribosome biogenesis can lead to nuclear retention of ribosomal subunits, ensuring only functional subunits are exported.
Ltv1, Enp1, and Pno1: These factors are associated with pre-40S particles and play roles in the nuclear export and cytoplasmic processing of the 40S subunit.

The nuclear export of ribosomal subunits is a crucial step in ribosome biogenesis and protein synthesis. The aforementioned players and mechanisms ensure that eukaryotic cells maintain the fidelity and efficiency of this process, optimizing cellular function and health.

tRNA structure monitoring

Only tRNAs with mature 5′ and 3′ ends are exported. 8 Transfer RNAs (tRNAs) are essential for protein synthesis, ensuring that amino acids are properly incorporated into the growing peptide chain. It's crucial for the cell to monitor tRNA maturation and only allow structurally mature tRNAs to be exported from the nucleus to the cytoplasm. Here are some of the key players and mechanisms involved in tRNA structure monitoring:

RNase P: This ribonucleoprotein complex processes the 5′ end of precursor tRNAs (pre-tRNAs). It ensures that tRNAs have mature 5′ ends before they undergo further processing and nuclear export.
RNase Z (or ELAC2 in humans): This endonuclease cleaves pre-tRNA at the 3′ end downstream of the discriminator nucleotide, generating mature 3′ termini.
CCA-adding enzyme: In eukaryotes, this enzyme post-transcriptionally adds a CCA sequence at the 3′ end of tRNAs, which is essential for tRNA function in protein synthesis.
tRNA modification enzymes: Several enzymes are involved in site-specific modifications of tRNA bases and sugar moieties. Examples include pseudouridine synthases, tRNA methyltransferases, and tRNA isopentenyltransferases. Properly modified tRNAs are critical for maintaining tRNA stability, structure, and function.
Los1/Exportin-t: In yeast, Los1 is the main export receptor for mature tRNAs, while in humans, the orthologous Exportin-t recognizes and binds to mature tRNAs, facilitating their export through the nuclear pore complex (NPC).
Nuclear Pore Complex (NPC): The tRNA export process occurs through the NPC. Only tRNAs that have undergone proper end processing and modifications are efficiently recognized and exported to the cytoplasm.
THO-TREX Complex: This complex plays a role in coupling transcription with tRNA nuclear export in yeast. Mutations affecting the complex can cause nuclear retention of tRNA.
La protein: Binds to the 3′ end of nascent pre-tRNA transcripts, protecting them from exonucleolytic degradation and ensuring correct end processing.
Mtr10: In yeast, this protein is involved in nuclear import of certain tRNA modification enzymes, ensuring a supply of enzymes in the nucleus for proper tRNA maturation.
TSEN Complex: This endonuclease complex is involved in tRNA splicing, specifically for intron-containing tRNAs. Proper splicing is a prerequisite for tRNA functionality.
Nucleocytoplasmic Quality Control: This mechanism ensures that only fully processed and matured tRNAs get exported to the cytoplasm. Misfolded or improperly processed tRNAs are retained in the nucleus for quality control.

Ensuring the structural integrity and proper processing of tRNAs is vital for accurate and efficient protein synthesis. The aforementioned players and mechanisms provide rigorous surveillance and quality control, ensuring the cellular protein-making machinery operates with precision.

tRNA synthesis quality control 

RTD and other mechanisms degrade hypomodified or mutated mature yeast tRNAs. 9 The synthesis and proper maturation of transfer RNAs (tRNAs) are essential for protein synthesis, as they act as intermediaries between mRNAs and amino acids. Quality control mechanisms are in place to ensure that only fully functional tRNAs participate in translation. Hypomodified or mutated tRNAs can compromise translation fidelity and thus are targeted for degradation. Here are some of the key players and mechanisms involved in tRNA synthesis quality control:

Rapid tRNA Decay (RTD) Pathway: This is a key mechanism in yeast to degrade hypomodified tRNAs, especially those lacking the m^5C modification at position 34. The RTD pathway targets such aberrant tRNAs for degradation to prevent them from participating in translation. Key proteins in this pathway include the exonuclease Rat1 and its associated protein Rai1.
Nuclear Exosome: This multi-subunit complex, prevalent in both the nucleus and cytoplasm, is responsible for the degradation of improperly processed tRNA precursors. Key components include the exoribonuclease RRP6 and the core exosome complex, which contains several ribonucleolytic subunits.
Trm7: This is a tRNA methyltransferase responsible for adding the m^5C modification at position 34. Mutations or malfunctions in Trm7 can lead to the production of hypomodified tRNAs, which are subsequently targeted by the RTD pathway.
Aminoacyl-tRNA Synthetases: These enzymes charge tRNAs with their corresponding amino acids. If a tRNA is mutated or improperly folded, it may not be recognized by its corresponding aminoacyl-tRNA synthetase, and thus, it remains uncharged and becomes a target for degradation.
Met22/Thi2: In yeast, this kinase is involved in signaling pathways that respond to tRNA damage or misprocessing. When activated, it can modulate tRNA synthesis and degradation pathways.
tRNA Nuclear-Cytoplasmic Trafficking: Properly folded and modified tRNAs are exported from the nucleus to the cytoplasm. tRNAs that are misfolded or bear mutations may be retained in the nucleus, preventing them from participating in translation.
Tod1/DXO: This exonuclease in yeast and mammals, respectively, can degrade certain aberrant tRNAs, especially those with 5′ or 3′ extensions.
TRAMP Complex: This complex assists the exosome in targeting aberrant tRNAs for degradation. It contains a poly(A) polymerase, a zinc-knuckle protein, and the helicase Mtr4.

Ensuring the accuracy and functionality of tRNAs is paramount for the fidelity of protein synthesis. The mentioned players and mechanisms provide rigorous surveillance of tRNA synthesis, ensuring that defective tRNAs are quickly recognized and removed from the translation machinery.

mRNA Cap Structure and Translation Regulation

Influence of mRNA cap structures on translation. The 5′ cap structure of eukaryotic mRNAs plays a pivotal role in various aspects of mRNA metabolism, including stability, splicing, transport, and, crucially, translation initiation. The cap is essential for efficient and accurate translation. Quality control and surveillance mechanisms ensure that mRNAs with proper cap structures are recruited for translation, while those with aberrant structures are targeted for degradation or other regulatory responses. Here are key players and mechanisms involved in monitoring and regulating mRNA cap structures:

eIF4F Complex: This multi-protein complex comprises eIF4E (which directly binds the cap structure), eIF4G, and eIF4A. It facilitates the recruitment of the 40S ribosomal subunit to the mRNA. Proper cap recognition by eIF4E ensures that only appropriately capped mRNAs are selected for translation.
Decapping Enzymes: DCP1 and DCP2 are core components of the decapping complex. They remove the 5' cap from mRNAs, marking them for degradation. Aberrant mRNAs with incorrect cap structures can be recognized and decapped, preventing their translation.
5' to 3' Exoribonucleases: Once the cap is removed, enzymes like Xrn1 degrade the mRNA from the 5' end, ensuring that improperly capped mRNAs do not accumulate in the cell.
CBC (Cap-Binding Complex): Comprising the proteins CBP80 and CBP20, CBC binds to the cap structure soon after transcription. While it aids in several mRNA processing steps, it also provides an initial quality control check for the cap structure.
Pan2-Pan3 and Ccr4-Not Complexes: These are cytoplasmic deadenylase complexes. Proper capping can influence the stability of mRNA by affecting deadenylation rates. mRNAs with aberrant cap structures might be more rapidly deadenylated and, subsequently, degraded.
NMD (Nonsense-Mediated Decay): While primarily a mechanism to detect and degrade mRNAs with premature stop codons, NMD can also be influenced by cap-binding proteins and, therefore, indirectly by the cap structure itself.
eIF4E Transporter (4E-T): This protein can sequester eIF4E, preventing it from binding to the cap structure. In doing so, 4E-T acts as a regulator ensuring that only mRNAs with appropriate cap structures are engaged by the translation machinery.
Phosphorylation of eIF4E: Kinases such as Mnk1/2 phosphorylate eIF4E. Phosphorylation can influence the affinity of eIF4E for the cap structure, providing an additional layer of regulation and quality control.

These systems work in tandem to ensure that only mRNAs with correct cap structures are efficiently translated, while those with aberrations are quickly identified and managed. This is crucial for cellular function, as inappropriate translation can have detrimental consequences for the cell.

References

Pre-translation Quality Control Extant in Both, Prokaryotes and Eukaryotes

tRNA Processing and Quality Control

tRNA Processing Mechanisms

Ensures tRNA molecules' correct maturation. tRNA molecules are critical components in protein synthesis, ensuring that amino acids are correctly incorporated into growing polypeptide chains based on the mRNA sequence. To function properly, precursor tRNA (pre-tRNA) transcripts undergo a series of maturation processes. Eukaryotic cells have established various quality control and surveillance mechanisms to ensure the proper processing and maturation of tRNAs. Here are some of the key players and mechanisms involved in tRNA processing:

Endonucleolytic Cleavage: After transcription, pre-tRNA molecules contain extra sequences at both their 5′ and 3′ ends. The RNase P complex is responsible for cleaving the 5′ leader sequence, while the RNase Z enzyme cleaves the 3′ trailer.
CCA Addition: Mature tRNAs have a CCA sequence at their 3′ end, which is essential for amino acid attachment. This sequence is added by the enzyme tRNA nucleotidyl transferase.
Intron Splicing: Some tRNAs contain introns that need to be removed. The tRNA splicing endonuclease complex recognizes and cleaves these introns. Subsequent ligation of the exons is performed by tRNA ligase.
Base Modifications: Several bases in tRNA molecules undergo chemical modifications, which are critical for tRNA function. Modification enzymes, like pseudouridine synthases and tRNA methyltransferases, introduce these changes.
TSEN Complex: A multi-protein complex responsible for recognizing and cleaving introns in certain pre-tRNAs.
La Protein: Binds to the 3' end of newly transcribed pre-tRNA molecules, protecting them from exonucleolytic degradation and aiding in their proper maturation.
RTD Pathway: Rapid tRNA Decay pathway in yeast ensures that hypomodified or mutated mature tRNAs are recognized and degraded, thus preventing their participation in translation.
TRAMP Complex: Works in conjunction with the exosome to degrade aberrant tRNA molecules in the nucleus.
tRNA Export Receptors: Proteins such as Los1 and exportin-t recognize mature tRNAs and facilitate their export from the nucleus to the cytoplasm, ensuring that only fully processed tRNAs participate in translation.
Aminoacyl-tRNA Synthetases: While primarily involved in charging tRNAs with their respective amino acids, these enzymes also provide a layer of quality control, as incorrectly processed tRNAs might not be efficiently or accurately aminoacylated.

These mechanisms and associated protein players work cohesively to ensure that tRNA molecules are correctly processed and matured. Proper tRNA maturation is crucial as these molecules play a direct role in the fidelity of protein synthesis, and any aberrations can have significant effects on cellular function.

tRNA Aminoacylation Quality Control 

Ensures tRNAs are charged with the right amino acids. tRNA aminoacylation, also known as tRNA charging, is a critical step in protein synthesis, where tRNAs are loaded with their corresponding amino acids. This process must be accurate, as mischarged tRNAs can lead to misincorporation of amino acids during translation, compromising the integrity of proteins. Eukaryotic cells have adopted several mechanisms to ensure the fidelity of this process. Here are the key players and mechanisms involved in tRNA aminoacylation quality control:

Aminoacyl-tRNA Synthetases (aaRSs): A family of enzymes responsible for attaching specific amino acids to their corresponding tRNAs. Each aaRS has a unique specificity for a particular amino acid and tRNA pair.
Editing Sites of aaRSs: Some aaRSs have built-in editing sites that can remove incorrectly attached amino acids from tRNAs, ensuring that only the right amino acid is attached to each tRNA.
Alanyl-tRNA Synthetase (AlaRS): While charging tRNA^Ala, this enzyme has an editing site that removes non-alanine amino acids, especially serine and glycine, which could be mistakenly attached.
Threonyl-tRNA Synthetase (ThrRS): Removes serine, which resembles threonine and can be erroneously attached to tRNA^Thr.
Valyl-tRNA Synthetase (ValRS): Has an editing site that removes threonine, ensuring only valine is attached to tRNA^Val.
Post-transfer Editing: For some aaRSs, mischarged tRNAs are released but are subsequently corrected by hydrolytic editing in a separate active site.
YbaK and ProXp-ala: These are trans-editing proteins found in some organisms that correct mischarged tRNAs when the aaRS fails to do so.
D-Tyr-tRNATyr Deacylase (DTD): Removes D-tyrosine, an unnatural amino acid, from tRNA^Tyr, ensuring that only the natural L-form is incorporated during protein synthesis.
ATP/AMP Ratio Sensing: The balance between ATP and AMP levels can influence the aminoacylation of tRNAs. A high ATP/AMP ratio encourages aminoacylation, while a low ratio can act as a checkpoint, ensuring energy sufficiency for translation.
Non-discriminating aaRSs: These enzymes can aminoacylate both canonical and non-canonical tRNA species, ensuring that the pool of charged tRNAs is maintained, especially under stress conditions.

The aforementioned mechanisms and proteins form a robust surveillance system, ensuring the accuracy of tRNA charging. Proper aminoacylation is central to the fidelity of protein synthesis, and ensuring this accuracy is paramount for the proper functioning and survival of the cell.

tRNA Anticodon Loop Modifications 

Modifications in the tRNA anticodon loop. The anticodon loop of tRNA is a critical region that interacts directly with the mRNA codon during protein translation, ensuring the correct insertion of amino acids into a nascent polypeptide. Modifications in the anticodon loop of tRNAs play a pivotal role in enhancing the accuracy and efficiency of this decoding process. Eukaryotic cells have evolved various mechanisms and players to introduce and maintain these modifications. Here are some key players and mechanisms involved in tRNA anticodon loop modifications:

Pseudouridine Synthases: These enzymes are responsible for converting uridine residues into pseudouridine (Ψ) in the anticodon loop. Pseudouridylation enhances tRNA stability and can affect codon recognition.
Cytidine Acetyltransferases: Enzymes like Tcd1 in yeast introduce ac4C modifications in the tRNA anticodon loop, influencing translation fidelity.
tRNA Uridine 5-oxyacetic acid methyltransferase (Trm9): In yeast, Trm9 adds a methoxycarbonylmethyl (mcm^5) group to certain uridines in the anticodon loop, enhancing the decoding capacity of tRNAs.
AlkB Homologs: These enzymes can reverse certain methylation events in the anticodon loop, acting as a repair mechanism for erroneous modifications.
Queuosine Modification: Queuine tRNA-ribosyltransferase introduces the queuosine (Q) modification at the wobble position of certain tRNAs, which can affect codon-anticodon pairing and translation fidelity.
Thiolation Enzymes: These enzymes introduce sulfur atoms into certain positions in the anticodon loop, enhancing tRNA stability and function. Examples include TtuA for tRNA thiolation in bacteria.
Methyltransferases: Several tRNA methyltransferases, such as TrmD and Trm5, modify the anticodon loop, affecting codon recognition and translation efficiency.
Anaerobic Modifications: Under anaerobic conditions, certain unique modifications can be introduced in the tRNA anticodon loop, such as the lysidine modification, which is crucial for decoding Ile in bacteria.
tRNA Modifying Enzymes and Stress: Environmental stress can influence the activity of tRNA modifying enzymes, leading to differential modification patterns that can affect translation dynamics.

The modifications in the anticodon loop of tRNAs are dynamic and can be influenced by various internal and external cellular factors. These modifications serve as a fine-tuning mechanism, ensuring the proper decoding of mRNA codons and enhancing the accuracy and efficiency of protein synthesis. Proper regulation and surveillance of these modifications are essential for cellular health and function.

tRNA Modification Surveillance 

Mechanisms identify and degrade incorrectly modified tRNAs. Post-transcriptional modifications are pivotal for the function of tRNAs, ensuring accurate and efficient protein translation. However, mis-modifications can lead to a loss of tRNA function or translation errors. Consequently, eukaryotic cells have developed multiple surveillance mechanisms to identify and degrade incorrectly modified tRNAs. Here are some of the key players and mechanisms involved in tRNA modification surveillance:

Rapid tRNA Decay (RTD): In yeast, this is a prominent mechanism for the degradation of hypomodified tRNAs, especially those lacking modifications that stabilize tRNA structure or aid in codon recognition. Factors like Rtt101, Mms1, and Mms22 are involved in this pathway.
Alkylation Repair Enzymes: Enzymes like AlkB can repair alkylated tRNAs, ensuring their modifications remain accurate for function.
Trm4: This tRNA methyltransferase modifies the wobble position of certain tRNAs. In its absence, these tRNAs are often subjected to degradation, indicating a surveillance role for such modifications.
NUFIP: In metazoans, this factor recognizes certain unmodified tRNAs and promotes their degradation, ensuring that only properly modified tRNAs participate in translation.
Metazoan CCA-adding Enzymes: These enzymes can discriminate between properly modified and hypomodified tRNAs, adding CCA only to correctly modified tRNAs. The absence of CCA on tRNAs often leads to their degradation.
ELAC2: A ribonuclease involved in tRNA maturation, it also plays a role in degrading improperly processed or modified tRNAs.
tRNA Nuclear Export: In eukaryotes, only properly processed and modified tRNAs are exported from the nucleus to the cytoplasm. This acts as a surveillance mechanism, ensuring that hypomodified tRNAs do not participate in translation.
Aminoacyl-tRNA Synthetases: While these enzymes charge tRNAs with their respective amino acids, they can also proofread and correct charging errors. Mis-modified tRNAs may have reduced affinity for their cognate aminoacyl-tRNA synthetases, leading to reduced charging and eventual degradation.
TRAMP Complex: Assists in the degradation of improperly processed or mis-modified tRNAs, working alongside the RNA exosome complex.
Discriminator Base Surveillance: The discriminator base, located just before the CCA sequence of tRNAs, plays a role in tRNA function. Aberrant modifications at this position can trigger tRNA degradation.

The above mechanisms highlight the importance of tRNA modifications for cellular functionality. By establishing robust surveillance systems, cells ensure translation accuracy and efficiency, vital for their health and adaptability.

Codon Optimization and tRNA Abundance

Maintains optimal tRNA levels for each codon. Proper translation efficiency and accuracy hinge on the harmonious interaction between codons in mRNA and their corresponding tRNA molecules. Codon optimization and the corresponding tRNA abundance ensure that translation is neither too slow nor prone to errors. Here are the key players and mechanisms that monitor and regulate this delicate balance:

Codon Bias: Codon usage is often biased towards specific codons for the same amino acid, especially in highly expressed genes. This bias often reflects the abundance of corresponding tRNAs, ensuring that these genes are translated efficiently.
tRNA Gene Copy Number: In eukaryotic genomes, the copy number of specific tRNA genes often correlates with the abundance of their corresponding tRNAs. This ensures that there are optimal levels of tRNAs available for the most frequently used codons.
Aminoacyl-tRNA Synthetases (AARSs): These enzymes charge tRNAs with their corresponding amino acids. The efficiency and accuracy of this process can influence the availability of charged tRNAs, affecting translation rates for specific codons.
tRNA Modification Enzymes: Post-transcriptional modifications in tRNA molecules, especially at the wobble position, can influence their pairing with codons. These modifications ensure that tRNAs can recognize and pair with multiple synonymous codons efficiently.
tRNA Decay Pathways: Mechanisms such as Rapid tRNA Decay (RTD) in yeast ensure that hypomodified or damaged tRNAs are promptly degraded, thereby maintaining a pool of functional tRNAs.
Ribosome Profiling: This technique provides insights into translation dynamics, including codon-pause sites. Pausing often correlates with rare codons and low-abundant tRNAs, helping cells identify and possibly rectify translation inefficiencies.
Adaptive Responses: In some organisms, stress or changing environmental conditions can lead to adjustments in tRNA abundance. For instance, upon amino acid starvation, certain tRNAs can be upregulated to maintain efficient translation of essential genes.
Codon Harmonization: This is an artificial method employed in biotechnology wherein codon usage of a gene is tailored to reflect the tRNA abundance of the host organism. It ensures efficient translation when expressing heterologous proteins.
tRNA-derived Fragments (tRFs): These are small RNA molecules derived from tRNAs. They can interfere with translation by displacing their corresponding full-length tRNAs, acting as a regulatory mechanism to modulate translation under certain conditions.
tRNA Sulfonation: Under oxidative stress, certain tRNAs undergo sulfonation, a modification that prevents their aminoacylation and thereby suppresses translation.

The orchestration between codon usage in mRNAs and tRNA abundance is a testament to the evolutionary optimization for translation efficiency and accuracy. Through these mechanisms and players, cells ensure that their proteome is synthesized in a timely and accurate manner, pivotal for their survival and function.

rRNA Synthesis, Processing, and Quality Control

rRNA Synthesis Error Check 

Surveillance mechanisms ensure that only correctly processed and modified rRNA is incorporated into ribosomes. Ribosomal RNA (rRNA) synthesis and its subsequent processing and modification are paramount for the proper function of ribosomes, the cellular machinery responsible for protein synthesis. To ensure the fidelity of this process, eukaryotic cells have established an array of surveillance mechanisms. Here are the pivotal players and mechanisms that oversee rRNA synthesis error check:

RNA Exosome Complex: A prominent player in rRNA quality control, this complex degrades aberrant rRNA molecules. Key subunits, like RRP44/Dis3 and RRP6, facilitate the degradation of improperly processed or unmodified rRNAs.
Nucleolar Surveillance: Erroneous pre-rRNA that hasn't undergone proper processing is sequestered in the nucleolus and is subsequently degraded. Nucleolar proteins such as NOB1 and XRN2 participate in the degradation of these flawed rRNA species.
5' ETS and ITS Checkpoints: The External and Internal Transcribed Spacers (ETS and ITS) are crucial for the accurate folding and processing of rRNA. Pre-rRNAs misprocessed or misfolded due to complications with these spacers are earmarked for degradation.
U3 snoRNA: This small nucleolar RNA enables the early cleavage events of pre-rRNA processing. Dysfunctions in the U3 snoRNA's interaction with pre-rRNA can initiate degradation pathways.
Small Nucleolar RNPs (snoRNPs): These particles are entrusted with the site-specific modification of rRNAs. rRNA molecules that are mis-modified, likely due to snoRNP issues, can be degraded. Guiding these modifications are snoRNAs, including C/D box and H/ACA box snoRNAs, with associated proteins such as fibrillarin and dyskerin facilitating the process.
TRAMP Complex: Acting in tandem with the exosome, this complex targets faulty RNAs for degradation. Integral components comprise a poly(A) polymerase, a zinc-knuckle protein, and the helicase Mtr4.
UTP-A, UTP-B, and UTP-C Complexes: These are embroiled in the initial steps of 18S rRNA processing. When these complexes falter, or the early processing steps face impediments, the rRNA precursors are held back and degraded.
Nop53p: This protein functions as a retention factor for 60S rRNA precursors that haven't been processed aptly, preventing their egress from the nucleolus.
Rrp5: A binding partner of pre-rRNA, Rrp5 aids its early processing steps. When these steps face complications due to Rrp5 malfunction, it can lead to rRNA degradation.

Through these meticulous mechanisms and players, eukaryotic cells ensure the rigorous oversight of rRNA synthesis and processing. This heightened quality control guarantees that ribosomes, vital for protein synthesis, operate with efficiency and precision.

Post-transcriptional Modifications of rRNA 

rRNA undergoes various modifications post-transcription essential for its function and ribosome assembly. After transcription, ribosomal RNA (rRNA) must undergo a plethora of modifications to ensure its proper function within the ribosome. These modifications are not only pivotal for the optimal performance of rRNA but also play a critical role in ribosome assembly. Let's delve into the key players and mechanisms involved in the post-transcriptional modifications of rRNA:

Small Nucleolar RNPs (snoRNPs): These are the main orchestrators of rRNA modifications. They are responsible for the majority of the methylations and pseudouridylations of rRNA. The two main classes, C/D box snoRNPs and H/ACA box snoRNPs, guide site-specific methylations and pseudouridylations, respectively.
Fibrillarin (FBL): A core protein component of the C/D box snoRNPs, FBL is the methyltransferase that catalyzes 2'-O-ribose methylation of rRNA.
Dyskerin (DKC1): An integral protein of the H/ACA box snoRNPs, dyskerin is responsible for the isomerization of uridine to pseudouridine in rRNA.
NOP58 and NOP56: These are additional protein components of the C/D box snoRNPs, essential for rRNA methylation.
NHP2 and GAR1: Proteins associated with the H/ACA box snoRNPs, they play a role in the pseudouridylation of rRNA.
Ribose Methylation: A modification where a methyl group is added to the ribose moiety of specific rRNA residues. C/D box snoRNAs guide this process.
Pseudouridylation: The conversion of uridine to pseudouridine, resulting in an isomerized base. This process is guided by H/ACA box snoRNAs.
Base Methylation: Certain bases in rRNA, particularly adenines and cytosines, can be directly methylated, enhancing rRNA stability and function.
RNA Helicase Prp43: Assists in the removal of snoRNPs after modification, ensuring that rRNA is free for subsequent processing and assembly steps.
Surveillance and Quality Control: Any mis-modified rRNA or mistakes in modification are targeted for degradation or repair. The RNA exosome complex, TRAMP complex, and other nucleolar proteins play a role in detecting and rectifying these errors.

These post-transcriptional modifications, and the proteins and RNAs involved in them, fine-tune the structure and function of rRNA. The modifications not only impart stability to the rRNA but also optimize its interactions within the ribosome, ensuring efficient and accurate protein synthesis.

Monitoring of Ribosomal RNA Modifications 

Ensures correct modifications in rRNA. Proper modifications of ribosomal RNA (rRNA) are essential for the optimal functionality of ribosomes. Monitoring systems have evolved to detect and correct anomalies, ensuring that only appropriately modified rRNA gets incorporated into ribosomes. Here are some of the key players and mechanisms involved in monitoring rRNA modifications:

snoRNA Surveillance: The major guiding molecules for rRNA modifications are small nucleolar RNAs (snoRNAs). Any snoRNA that fails to accumulate or mislocalizes can lead to aberrant rRNA modifications. The system ensures that snoRNAs guide modifications at the correct sites.
RNA Exosome Complex: Aberrantly modified rRNA, if undetected, can compromise ribosome function. The RNA exosome, especially components like RRP44/Dis3 and RRP6, targets mis-modified rRNA for degradation, maintaining the pool's integrity.
Nucleolar Quality Control: The nucleolus, the primary site for rRNA processing and modification, also serves as a quality checkpoint. Mis-modified rRNAs are retained in the nucleolus and subjected to degradation or repair.
UTP-B Complex: This complex is associated with early pre-rRNA processing and modification. It has been proposed that UTP-B might be involved in monitoring the correct modification of the 18S rRNA.
Nop53p: This nucleolar protein acts as a checkpoint for rRNA modifications, especially in the context of the 60S subunit. It binds to rRNA and ensures that only correctly modified rRNA gets incorporated into the maturing ribosomal subunit.
Grc3 and Las1: These proteins are crucial for the endonucleolytic cleavage at specific sites in pre-rRNA. They can also act as checkpoints to monitor correct rRNA modifications, especially around those cleavage sites.
Methyltransferases and Pseudouridine Synthases: The enzymes directly responsible for methylation and pseudouridylation of rRNA, respectively, also have inherent quality control roles. Their specificity and binding mechanisms ensure that modifications occur at the right positions.
TRAMP Complex: Similar to its role in rRNA synthesis error checks, the TRAMP complex, with its components like Mtr4 helicase, assists the exosome in degrading aberrant rRNAs that might have undergone incorrect modifications.
snoRNP Biogenesis Factors: Proteins such as Nop58, Nop56, Fibrillarin, and Nhp2, which are involved in the assembly and function of snoRNPs, indirectly ensure the correct rRNA modification by maintaining the integrity and functionality of snoRNPs.
Rrp5: Beyond its roles in rRNA synthesis error checks, Rrp5 also monitors rRNA modifications, ensuring the correct and timely incorporation of modified rRNA into ribosomes.

The intricate network of proteins, RNAs, and complexes ensures that the rRNA modifications are accurate, and any errors are swiftly addressed. This monitoring is crucial for the production of functional ribosomes, which, in turn, guarantees effective and accurate protein synthesis.

Ribosomal RNA Cleavage and Processing

Precursor rRNA molecules undergo cleavage processes to produce the mature rRNAs required for ribosome assembly. The processing of precursor ribosomal RNA (pre-rRNA) molecules to generate mature rRNAs is a crucial step in ribosome biogenesis. This intricate pathway involves a series of endonucleolytic and exonucleolytic cleavage events that ensure the proper formation of functional ribosomal subunits. Here are the key players and mechanisms involved in rRNA cleavage and processing, with a particular focus on error check and quality control:

Endonucleases and Exonucleases: Key enzymes like RNase MRP, RNase III, Xrn1, and the RNA exosome complex are involved in the cleavage and trimming of pre-rRNA transcripts. Their specificity ensures that cuts are made at the appropriate positions, generating the mature rRNA species.
5' ETS and ITS Checkpoints: The External and Internal Transcribed Spacers (ETS and ITS) not only act as guide markers for rRNA cleavage but also serve as checkpoints. Misprocessed pre-rRNAs due to issues with these spacers are targeted for degradation.
U3 snoRNA: This snoRNA molecule facilitates early cleavage events in pre-rRNA processing. Proper interaction between U3 snoRNA and pre-rRNA is essential for accurate processing. Any malfunction in this interaction can initiate degradation pathways.
Small Nucleolar RNPs (snoRNPs): Alongside their role in rRNA modification, snoRNPs, especially the box C/D and box H/ACA snoRNPs, guide cleavage and processing events. Any mishap or malfunction can lead to aberrant rRNA species which are then targeted for degradation.
RNA Exosome Complex: This multi-protein complex, especially with components like RRP44/Dis3 and RRP6, plays a pivotal role in the degradation of improperly processed pre-rRNAs, ensuring the quality of mature rRNAs.
TRAMP Complex: Acting in synergy with the exosome, this complex identifies aberrant rRNA species and aids in their degradation, maintaining the quality of rRNA processing.
Nop53p: This protein ensures the correct processing of the 60S rRNA precursors and prevents the export of any improperly processed species from the nucleolus.
Rrp5: Binding to pre-rRNA, Rrp5 assists in early processing steps. Any malfunction or hindrance in these steps can result in rRNA degradation pathways being initiated.
Nop1, Nop56, and Nop58: These proteins, involved in snoRNP biogenesis, also play roles in pre-rRNA cleavage. They ensure that cleavage events are synchronized with rRNA modifications.
UTP-A, UTP-B, and UTP-C Complexes: These complexes are integral to the early steps of pre-rRNA processing. If initial processing events are hindered, rRNA precursors can be degraded.

The comprehensive set of factors and mechanisms ensures that the pre-rRNA undergoes accurate processing to generate mature rRNAs, the core components of ribosomes. Quality control during this phase is vital as any aberrancy can severely compromise protein synthesis.

Ribosome Biogenesis, Synthesis, and Quality Control

Ribosome Synthesis in Response to Mechanical Stress

Influence of mechanical forces on ribosome synthesis. Mechanical stresses, arising from various external or internal factors, can influence cellular functions profoundly. These forces can particularly affect the transcriptional, translational, and post-translational machineries. Ribosome biogenesis, a pivotal cellular process for protein synthesis, is not exempt from the effects of mechanical stress. Here are some of the key players and mechanisms involved in the regulation of ribosome synthesis in response to mechanical stress, emphasizing error checks, quality control, and repair mechanisms:

Mechanical Stress Sensors: These proteins or complexes sense changes in the mechanical environment of the cell. Examples include stretch-activated ion channels and integrins. Activation of these sensors can trigger signaling pathways that influence ribosome synthesis.
mTOR Pathway: The mechanistic target of rapamycin (mTOR) is a central integrator of growth signals and cellular energy status. Mechanical stress can modulate mTOR activity, which in turn, affects ribosomal RNA (rRNA) transcription and ribosome assembly.
Ribosomal Stress Response: In response to mechanical forces, certain ribosomal proteins may become unbound from the nucleolus, leading to the activation of p53, a tumor suppressor protein. p53 can halt cell cycle progression and influence rRNA transcription, slowing down ribosome synthesis.
RNA Polymerase I: This enzyme is responsible for transcribing the large rRNA precursor. Mechanical stress may impact its activity directly or via regulatory factors, thus influencing the rate of ribosome synthesis.
Nucleolar Remodeling: The nucleolus, where ribosome biogenesis primarily occurs, may undergo structural and functional changes in response to mechanical stress. This remodeling can affect the synthesis and assembly of ribosomal components.
ATF4 Stress Response Pathway: The activating transcription factor 4 (ATF4) gets activated under various stress conditions, including mechanical stress. ATF4 can adjust the transcription of genes associated with ribosome biogenesis.
Quality Control Mechanisms: Mechanical stress may lead to errors in ribosome assembly. Surveillance mechanisms, like the RNA exosome complex and the TRAMP complex, may be upregulated or become more active to ensure the degradation of improperly processed or assembled ribosomal components.
RACK1: The Receptor for Activated C Kinase 1 (RACK1) is a ribosomal protein that can sense and respond to mechanical cues. It may influence the translation of specific mRNAs under mechanical stress conditions.
Heat Shock Proteins (HSPs): Under mechanical stress, cells may produce HSPs, which act as molecular chaperones. These proteins may assist in the proper folding and assembly of ribosomal proteins and rRNA, ensuring the integrity of newly synthesized ribosomes.
Autophagy: Extended mechanical stress may lead to cellular damage, including damage to ribosomes. Autophagy, a cellular recycling mechanism, may be activated to degrade and recycle damaged ribosomal components.

The cellular response to mechanical stress is multifaceted, with ribosome biogenesis being a critical target. A balanced synthesis of ribosomes under such conditions ensures that cells can effectively manage stress while maintaining the essential protein synthesis machinery.

Ribosome Synthesis in Response to Chemical Stress

Influence of chemicals on ribosome synthesis. Chemical stressors, which can range from toxins and drugs to changes in nutrient availability, can perturb cellular homeostasis and influence fundamental processes like ribosome biogenesis. Ribosome synthesis, vital for protein translation, can be modulated or even halted in response to certain chemical agents. Here are some of the key players and mechanisms involved in the regulation of ribosome synthesis under chemical stress, emphasizing error checks, quality control, and repair mechanisms:

mTOR Pathway: The mechanistic target of rapamycin (mTOR) is a primary regulator of cell growth and metabolism. Chemical agents that affect cellular energy status or growth signals can modulate mTOR activity, influencing ribosomal RNA (rRNA) transcription and ribosome assembly.
p53 Activation: Many genotoxic chemicals activate the tumor suppressor protein p53. Once activated, p53 can repress the transcription of ribosomal genes, slow down rRNA processing, and induce cell cycle arrest or apoptosis.
RNA Polymerase I Inhibition: Certain chemicals directly inhibit RNA Polymerase I, responsible for transcribing the precursor of large rRNAs. This leads to reduced rRNA synthesis and can disrupt ribosome biogenesis.
Nucleolar Stress Response: Chemical stressors can lead to nucleolar disruption, affecting ribosome synthesis. This can result in the release of ribosomal proteins, which then activate stress response pathways, such as those mediated by p53 or MDM2.
Heat Shock Proteins (HSPs): Chemical stress can lead to protein misfolding. In response, cells often upregulate HSPs, which act as molecular chaperones to assist in the proper folding of ribosomal proteins and rRNA.
RNA Modification Enzymes: Chemical stress may affect enzymes responsible for rRNA modifications. Alterations in these modifications can disrupt ribosome function, necessitating quality control mechanisms to degrade faulty ribosomes.
RNA Exosome Complex and TRAMP: These surveillance mechanisms may be more active under chemical stress, ensuring the degradation of improperly processed or assembled rRNA molecules.
Amino Acid Starvation: Chemicals that disrupt amino acid metabolism can lead to amino acid scarcity. This triggers the GCN2 pathway, which phosphorylates the eukaryotic initiation factor 2α (eIF2α), leading to global translation repression and selective translation of stress response genes.
Ribosome-Targeting Antibiotics: Many antibiotics target bacterial ribosomes. While these typically affect prokaryotic cells, prolonged exposure or high doses might indirectly affect eukaryotic ribosome biogenesis.
Autophagy: Certain chemicals can induce cellular damage, including damage to ribosomes. Autophagy might be activated to degrade and recycle damaged ribosomal components.
Oxidative Stress Response: Reactive oxygen species (ROS)-generating chemicals can damage rRNA, ribosomal proteins, and associated factors. Cells counteract this through antioxidants and DNA/RNA repair mechanisms to restore ribosome biogenesis.

Chemical stressors can significantly challenge ribosome synthesis and function. Through various mechanisms, cells strive to ensure that ribosome biogenesis continues efficiently, or it is temporarily halted to repair and restore cellular homeostasis.

Ribosome Biogenesis and Cell Cycle Regulation 

Mechanisms that coordinate ribosome synthesis with cell growth and division cycle.  Ribosome biogenesis and cell cycle progression are intertwined processes, ensuring that protein synthesis capacity matches the needs of a growing and dividing cell. Understanding how ribosome synthesis is coordinated with cell growth and division is fundamental to appreciating cellular homeostasis and proliferation. Here are some of the pivotal players and mechanisms that coordinate ribosome biogenesis with the cell cycle, with a focus on error checks, quality control, and repair mechanisms:

mTOR Pathway: A primary regulator of cell growth, metabolism, and ribosome biogenesis. It senses cellular nutrient levels and growth signals. When conditions are favorable, mTOR activation promotes rRNA transcription and ribosome assembly, while simultaneously promoting cell cycle progression.
p53 Pathway: This tumor suppressor plays a dual role. In response to DNA damage or ribosomal stress, p53 can halt cell cycle progression, allowing for repair mechanisms to act. Concurrently, p53 can repress rRNA transcription, thereby slowing ribosome biogenesis.
Rb-E2F Pathway: Retinoblastoma protein (Rb) binds and inhibits E2F transcription factors in quiescent cells. Upon cell cycle entry, Rb is phosphorylated and releases E2F, which then activates genes essential for DNA synthesis and ribosome biogenesis.
Nucleolar Size and Structure: The nucleolus, where ribosome synthesis occurs, changes in size and structure based on the cell's ribosome production rate, which in turn reflects the cell's metabolic activity and cell cycle stage.
Nucleolar Surveillance: Any disruption in ribosome biogenesis can lead to nucleolar stress. This stress can trigger cell cycle arrest, allowing the cell to resolve the ribosomal issues before proceeding.
Cyclin-Dependent Kinases (CDKs): These regulate cell cycle progression. CDK activity can influence ribosome biogenesis, particularly through phosphorylation events that impact RNA Polymerase I activity and rRNA processing.
Pol I and Pol III Transcription: The transcription of rRNA by RNA Polymerase I and tRNA by RNA Polymerase III is coordinated with the cell cycle, ensuring that ribosomal components are available in sync with cellular growth demands.
Ribosome-Associated Checkpoints: In yeast, specific checkpoints monitor ribosome biogenesis. For instance, if 60S subunit synthesis is impaired, the cell cycle can be arrested to allow for correction.
Nucleostemin: Found primarily in stem cells and cancer cells, this nucleolar protein links ribosome biogenesis to cell cycle progression. Its exact role remains a topic of study, but it's believed to coordinate ribosomal RNA synthesis with cell proliferation.
Ribosomal Proteins and Cell Cycle: Some ribosomal proteins, when free and not incorporated into ribosomes, can interact with the MDM2 ubiquitin ligase, impacting p53 stability and thereby influencing the cell cycle.
Autophagy and Proteostasis: Cells ensure proper protein homeostasis. If ribosome biogenesis is perturbed, cells might upregulate autophagy to degrade and recycle damaged ribosomal components, thereby preventing errors during cell division.

Ribosome biogenesis and the cell cycle are tightly regulated processes that must be in harmony for cell survival and proper function. Various checkpoints and mechanisms ensure this coordination, with disruptions potentially leading to diseases, including cancer.

Monitoring of Ribosome Biogenesis in the Nucleoplasm

Processes outside of the nucleolus that ensure the correct maturation of ribosomal components in eukaryotes.  Outside of the nucleolus, the nucleoplasm also plays a crucial role in ensuring the accurate maturation of ribosomal components in eukaryotic cells. The nucleoplasm harbors specific checkpoints and mechanisms to regulate, monitor, and correct errors associated with ribosome biogenesis. Below are key players and mechanisms engaged in this quality control process:

Nucleoplasmic Reticulum (NR): An invagination of the nuclear envelope, the NR is thought to provide a compartmentalized environment in the nucleoplasm for processes like calcium signaling and potentially ribosome assembly.
Exportin 1 (XPO1/CRM1): A primary nuclear export receptor responsible for the transport of many ribosomal components from the nucleus to the cytoplasm. XPO1 ensures that only properly assembled ribosomal subunits or those in the correct assembly state are exported.
Nucleophosmin (NPM1/B23): While primarily associated with the nucleolus, this protein can shuttle between the nucleolus and nucleoplasm. It plays roles in ribosome assembly and transport of ribosomal components.
RAN-GTPase System: Essential for the transport of ribosomal subunits across the nuclear envelope. The RAN-GTP/GDP gradient ensures that only mature ribosomal subunits are translocated to the cytoplasm.
TREX-2 Complex: Involved in the mRNA and ribosomal RNA export. Ensures efficient and selective export of RNA components of the ribosome.
Quality Control and Retention Mechanisms: Aberrant ribosomal subunits or assembly intermediates are detected and retained within the nucleoplasm, preventing their export. This retention ensures that only functional ribosomes reach the cytoplasm.
Ribosomal Protein Ubiquitination: In situations where ribosomal proteins are in excess or not correctly incorporated into ribosomes, they can undergo ubiquitination, marking them for degradation and thus maintaining a balanced pool of ribosomal components.
Ribosome-Associated Chaperones: Such as Hsp90 and its co-chaperones, assist in the proper folding of ribosomal proteins in the nucleoplasm and facilitate their correct assembly.
Pumilio Homology Domain-Containing Protein (PUM3): This protein has been associated with the quality control of rRNA in the nucleoplasm, ensuring only correctly processed rRNA proceeds to the next stages of ribosome assembly.
Perturbations and Stress Responses: The cell can sense defects in ribosome biogenesis in the nucleoplasm. Such sensing can lead to responses like nucleolar stress or p53 activation, ensuring that problematic ribosomal components are not incorporated into functional ribosomes.

The intricate processes in the nucleoplasm ensure that ribosome biogenesis is error-free. Any discrepancy or aberration detected activates quality control mechanisms to rectify or degrade the faulty components, ensuring optimal ribosome functionality in the cellular translational machinery.

Monitoring of Ribosome Biogenesis in the Nucleolus

The nucleolus, where ribosomes are synthesized, monitors correct ribosome biogenesis. The nucleolus, a prominent subnuclear organelle responsible for ribosome biogenesis, plays a pivotal role in ensuring the accurate and efficient assembly of ribosomal subunits in eukaryotic cells. Within the nucleolus, various checkpoints and mechanisms are in place to oversee, regulate, and maintain the quality of ribosome biogenesis. Here are the key players and mechanisms engaged in this quality control process within the nucleolus:

Small Nucleolar RNAs (snoRNAs): These non-coding RNAs guide the site-specific modifications of ribosomal RNA (rRNA) molecules. SnoRNAs, such as C/D box and H/ACA box snoRNAs, ensure that rRNAs are correctly modified during ribosome biogenesis.
Nucleolar Surveillance Mechanisms: Any rRNA that fails to undergo proper processing or modification is subjected to surveillance mechanisms within the nucleolus. Nucleolar proteins like NOB1 and XRN2 are involved in identifying and degrading aberrant rRNA species.
Assembly Factor Proteins: Proteins like nucleophosmin (NPM1/B23) and nucleolin (NCL) assist in the assembly and maturation of ribosomal subunits within the nucleolus. They ensure that ribosomal components are correctly folded and integrated into the growing ribosomal subunits.
Nucleolar Retention Factors: Aberrant ribosomal subunits or intermediates that do not meet quality standards are retained within the nucleolus. These retention factors prevent the export of faulty ribosomal components, ensuring that only functional ribosomal subunits reach the cytoplasm.
Quality Control and Surveillance Mechanisms: Various mechanisms exist to detect errors in ribosome biogenesis, including problems in rRNA processing, modifications, or protein incorporation. These mechanisms activate quality control responses to rectify or degrade problematic components.
RNA Polymerase I (Pol I) Transcription Monitoring: The regulation of Pol I transcription ensures that the proper amounts of rRNA are synthesized for ribosome biogenesis. Any discrepancies in Pol I activity can lead to adjustments to maintain ribosome biogenesis quality.
Ribosomal Protein Chaperones: Chaperone proteins like Hsp90 and its co-chaperones assist in the proper folding of ribosomal proteins within the nucleolus, ensuring they are correctly incorporated into ribosomal subunits.
Ribosomal Protein Quality Control: Proteins that fail to integrate correctly into ribosomal subunits can undergo quality control processes, including ubiquitination, to mark them for degradation. This maintains a pool of high-quality ribosomal components.
Nucleolar Stress Responses: The nucleolus can sense disruptions in ribosome biogenesis and activate stress responses, such as the nucleolar stress response or p53-mediated pathways, to address errors and maintain ribosome quality.

These mechanisms within the nucleolus collectively ensure that ribosome biogenesis proceeds accurately and efficiently. Any deviations or errors are promptly detected and rectified, leading to the production of high-quality ribosomes crucial for cellular protein synthesis.

mRNA Processing and Interaction with Ribosomes

mRNA Structure and Ribosome Interaction

Ensures ribosomes can effectively navigate mRNA secondary structures. The interaction between ribosomes and mRNA structures is crucial for efficient translation. To ensure ribosomes can effectively navigate mRNA secondary structures, various quality control and regulatory mechanisms are in place. Here are some of the key players and mechanisms involved in this process:

RNA Helicases: RNA helicases, such as eIF4A, play a vital role in unwinding complex mRNA secondary structures. They use energy from ATP hydrolysis to disrupt stable RNA duplexes, allowing ribosomes to access the mRNA coding region.
Translation Initiation Factors: Initiation factors like eIF4E, eIF4G, and eIF4A facilitate the assembly of the 43S pre-initiation complex (comprising the small ribosomal subunit and associated factors) at the mRNA's 5' cap structure. This helps position the ribosome correctly at the mRNA's start codon, bypassing inhibitory mRNA structures.
Ribosome Pausing and Stalling Detection: Ribosomes have intrinsic mechanisms to detect pausing or stalling caused by mRNA secondary structures or rare codon sequences. This detection triggers various quality control pathways, including the Ribosome-Associated Quality Control (RQC) system, which targets and resolves stalled ribosomes.
RNA-Binding Proteins: RNA-binding proteins, such as RNA helicases, poly(A)-binding proteins (PABP), and various RNA-binding proteins, interact with mRNA to modulate its structure and accessibility to ribosomes. They help in mRNA remodeling to facilitate ribosome binding.
Secondary Structure Prediction Algorithms: Computational tools and algorithms are used to predict potential mRNA secondary structures. These predictions aid researchers in understanding how mRNA structures might affect ribosome binding and translation efficiency.
mRNA Cap Structure: The mRNA cap structure at the 5' end plays a role in promoting ribosome binding and translation initiation. Proteins like eIF4E recognize and bind to the cap, facilitating ribosome recruitment.
Ribosomal RNA (rRNA) Modifications: Modifications in the rRNA components of the ribosome can influence ribosome-mRNA interactions. For example, pseudouridylation of rRNA can affect ribosome binding to mRNA.
Translational Control Elements: Specific RNA sequences, such as internal ribosome entry sites (IRES) and upstream open reading frames (uORFs), can modulate ribosome recruitment and bypass mRNA secondary structures during translation initiation.
RNA Remodeling Complexes: Complexes like the CCR4-CAF1-Not complex can interact with mRNA and influence its structure, impacting ribosome interactions.
mRNA Circularization: Circularization of mRNA through interactions between the 5' cap-binding complex and the poly(A) tail-binding proteins enhances ribosome binding and translation efficiency.
Nonsense-Mediated Decay (NMD): NMD is a surveillance mechanism that degrades mRNAs with premature stop codons. It ensures that ribosomes do not engage with and attempt to translate aberrant mRNA sequences.

These players and mechanisms collectively contribute to the quality control of mRNA structures and ribosome interactions, ensuring that ribosomes can effectively navigate complex mRNA secondary structures and initiate translation with precision and fidelity.

mRNA Surveillance via Ribosome Profiling

Techniques that provide insights into ribosome-mRNA interactions, revealing potential errors or irregularities. Ribosome profiling, also known as Ribo-seq, is a powerful technique that provides insights into ribosome-mRNA interactions, revealing potential errors or irregularities in translation. This method involves the deep sequencing of ribosome-protected mRNA fragments, allowing researchers to analyze the positions of ribosomes on mRNA transcripts. Here's how ribosome profiling serves as a quality control mechanism:

Ribosome Positioning Analysis: Ribosome profiling enables the precise mapping of ribosome positions along mRNA transcripts. By analyzing ribosome densities at each codon, researchers can identify ribosome pausing, stalling, or frameshifting events, which may indicate translation errors or defects.
Identification of Translational Errors: Ribosome profiling can reveal instances where ribosomes encounter premature stop codons (nonsense codons) or frameshift events. These errors can lead to the production of truncated or misfolded proteins. Detecting such errors helps in quality control and may trigger mRNA degradation through nonsense-mediated decay (NMD).
Quantification of Translation Efficiency: Ribosome profiling allows for the quantification of ribosome occupancy on each mRNA, providing insights into translation efficiency. Deviations from the expected translation rates may indicate regulatory mechanisms or defects in translation.
Detection of Ribosome Pausing and Stalling: Ribosomes may pause or stall during translation due to mRNA secondary structures, rare codons, or other factors. Ribosome profiling helps identify such events, shedding light on potential translation errors or regulatory checkpoints.
Quality Control During Stress Conditions: Ribosome profiling can be used to monitor translation dynamics under stress conditions. Cells may adapt translation to prioritize specific genes during stress responses, and ribosome profiling reveals these changes.
Translational Frame Analysis: In cases where ribosomes shift frames during translation, ribosome profiling can detect the position and frequency of frame-shifting events. Such errors can result in the production of incorrect protein sequences.
Identification of Ribosome Queuing: Ribosome profiling can uncover instances where ribosomes queue up on an mRNA due to various factors. This queuing may indicate issues with translation elongation and can be a target for quality control mechanisms.
Monitoring of Ribosome Recycling: Ribosome profiling can provide insights into the recycling of ribosomes after translation termination. Detecting stalled ribosomes and their fate (recycling or degradation) contributes to quality control.
Comparative Analyses: Ribosome profiling data from different experimental conditions or cell types can be compared to identify variations in translation patterns, uncovering potential defects or regulatory mechanisms.

Overall, ribosome profiling serves as a valuable tool for monitoring mRNA translation and ensuring the fidelity and efficiency of protein synthesis. It allows researchers to detect translational errors, ribosome behavior, and regulatory mechanisms, contributing to the quality control of protein production in cells.

mRNA Secondary Structure Recognition

Mechanisms that ensure ribosomes navigate and translate through complex mRNA secondary structures. Ensuring ribosomes can effectively navigate and translate through complex mRNA secondary structures is crucial for accurate and efficient protein synthesis. To achieve this, cells employ various mechanisms and players that recognize, unwind, or overcome intricate mRNA structures. These mechanisms serve as quality control measures to prevent translation errors and ensure the fidelity of protein synthesis. Here are key players involved in mRNA secondary structure recognition:

RNA Helicases: RNA helicases are enzymes that unwind RNA secondary structures. They play a critical role in ensuring ribosomes can access the mRNA coding region without impediments. Helicases like eIF4A in eukaryotes and RhlB in bacteria actively unwind structured mRNA regions during translation initiation.
Initiation Factors: Initiation factors, such as eIF4F in eukaryotes and IF1 in bacteria, assist in recruiting ribosomes to the mRNA's 5' cap and scanning for the start codon. They help ensure that ribosomes can initiate translation accurately, even in the presence of structured mRNA regions.
Ribosomal RNA (rRNA) Base Pairing: The rRNA components of the ribosome can form base pairs with mRNA sequences, helping to stabilize mRNA on the ribosome and facilitate translation through structured regions. These base-pairing interactions can help prevent ribosome stalling.
Translational GTPases: Translational GTPases, such as EF-G in bacteria and eEF1A in eukaryotes, participate in the translocation step of translation. They help move the ribosome along the mRNA, overcoming structural obstacles and ensuring proper codon-anticodon pairing.
Ribosome Pausing and Rescue Factors: When ribosomes encounter strong secondary structures in the mRNA that impede translation, ribosome pausing can occur. Specialized factors, such as RQC (Ribosome Quality Control) in eukaryotes and rescue factors like tmRNA-SmpB in bacteria, recognize stalled ribosomes and facilitate their release or degradation, preventing translational errors.
RNA-Binding Proteins: Various RNA-binding proteins, such as RNA chaperones, can bind to structured mRNA regions and remodel or stabilize the mRNA secondary structure. This helps ribosomes navigate through challenging regions.
Upstream Open Reading Frames (uORFs): In eukaryotes, uORFs are short coding sequences in the 5' untranslated region (UTR) of mRNA that can affect ribosome scanning and translation initiation. They are often involved in regulatory mechanisms that sense and respond to mRNA secondary structures.
Non-Coding RNAs (ncRNAs): Certain ncRNAs, like small regulatory RNAs, can interact with structured mRNA regions and influence translation. They may act as guides or facilitators in navigating complex secondary structures.
ATP-Dependent RNA Remodeling Complexes: ATP-dependent RNA remodeling complexes, such as the ribosome-associated RQC complex in eukaryotes, utilize energy to disrupt structured mRNA regions and facilitate translation.
tRNA Modifications: Some tRNA modifications play a role in enhancing the decoding of structured mRNA regions. Modified tRNAs can help maintain translation accuracy under challenging conditions.

These mechanisms collectively contribute to mRNA secondary structure recognition and enable ribosomes to effectively decode the genetic information in mRNAs, even when faced with intricate structural barriers. This quality control ensures that proteins are synthesized accurately and efficiently in cells.

Ribosome Synthesis Responses to External Signals

Ribosome Synthesis in Response to Hormonal Signals 

Hormones can influence ribosome synthesis in eukaryotic cells. Hormones play a crucial role in regulating various cellular processes, including ribosome synthesis, in eukaryotic cells. The influence of hormonal signals on ribosome biogenesis is a vital aspect of cellular control. Here, we focus on the players and mechanisms involved in error check, quality control, repair, and discarding/recycling of ribosomal components in response to hormonal signals:

Hormone Receptors: Cells possess specific receptors for various hormones. When hormones bind to their respective receptors, it initiates signaling cascades that can impact ribosome synthesis.
Transcription Factors: Hormone-receptor complexes can activate or repress transcription factors that regulate the expression of genes involved in ribosome biogenesis. These factors help ensure the correct synthesis of ribosomal RNA (rRNA) and ribosomal proteins.
Upstream Binding Factors: Upstream binding factors, like UBF (Upstream Binding Factor) in eukaryotes, are crucial for rRNA transcription. Hormonal signaling can modulate the activity of these factors, influencing rRNA synthesis.
RNA Polymerase I (Pol I): Pol I is responsible for transcribing rRNA genes. Hormone-induced signaling can regulate the activity of Pol I and control rRNA production.
mTOR Pathway: The mTOR (mammalian target of rapamycin) pathway is a central regulator of cellular growth and ribosome biogenesis in response to hormonal signals. mTOR integrates various inputs, including hormonal cues, to control the synthesis of ribosomal components.
Quality Control Mechanisms: Hormones can influence the activity of quality control mechanisms that monitor ribosome assembly. For instance, hormonal signals may affect the function of RNA surveillance pathways that ensure only properly processed rRNA is incorporated into ribosomes.
Ribosome Assembly Factors: Hormonal signals can modulate the expression or activity of ribosome assembly factors, which assist in the correct folding and assembly of ribosomal subunits.
Nucleolar Surveillance: The nucleolus, where ribosomes are synthesized, is a key site for hormonal regulation of ribosome biogenesis. Hormones can impact nucleolar processes, including the surveillance of ribosomal components for quality control.
Ribosome Recycling: Hormonal signals can influence the recycling of ribosomal subunits and components. For example, hormones may affect the activity of ribosome-associated chaperones and recycling factors.
RNA Modifications: Some hormones may influence the post-transcriptional modifications of rRNA, ensuring that ribosomes maintain their functionality.
Protein Degradation Pathways: Hormonal signals can regulate protein degradation pathways that target defective ribosomal proteins or misassembled ribosomal subunits.
Cellular Stress Responses: Hormones can activate cellular stress responses that impact ribosome biogenesis. These responses may involve the inhibition or enhancement of ribosome synthesis, depending on the cellular context.
Coordination with Growth and Development: Hormonal signals often play a central role in coordinating ribosome synthesis with the growth and development of an organism or tissue.

Hormonal regulation of ribosome synthesis is a complex and highly orchestrated process. It ensures that ribosome production aligns with the physiological needs of the cell and the organism as a whole. These mechanisms help maintain cellular homeostasis and ensure that the ribosomes produced are functional and capable of supporting protein synthesis in response to hormonal signals.

Regulation of Ribosomal RNA Transcription

Mechanisms that adjust the rate of rRNA transcription according to cellular needs. The regulation of ribosomal RNA (rRNA) transcription is a crucial process that adjusts the rate of rRNA synthesis according to cellular needs. Here, we focus on the players and mechanisms involved in error check, quality control, repair, and discarding/recycling of ribosomal components during the regulation of rRNA transcription:

RNA Polymerase I (Pol I): RNA Polymerase I is responsible for transcribing rRNA genes. It plays a central role in the regulation of rRNA transcription. Pol I activity is finely tuned to ensure the appropriate rate of rRNA synthesis.
Transcription Factors: Various transcription factors regulate Pol I activity. These factors, including SL1 (selectivity factor 1), UBF (Upstream Binding Factor), and TIF-IA (Transcription Initiation Factor IA), are involved in promoter recognition and the initiation of rRNA transcription. They help control the error check during the early stages of transcription.
Epigenetic Regulation: Epigenetic modifications, such as DNA methylation and histone modifications, can influence the accessibility of rRNA genes for transcription. These modifications are part of the quality control mechanisms that ensure proper rRNA transcription.
Ribosomal RNA Processing Factors: Factors involved in rRNA processing and modification can also impact rRNA transcription. Ensuring correct processing is essential for the production of functional ribosomes.
Nucleolar Surveillance: The nucleolus, where rRNA transcription and processing predominantly occur, monitors the correctness of these processes. Aberrant rRNA transcription can trigger quality control mechanisms within the nucleolus.
Cellular Signaling Pathways: Cellular signaling pathways, influenced by various factors including nutrient availability and stress responses, can modulate the rate of rRNA transcription. These pathways serve as checkpoints to ensure that ribosome production aligns with cellular conditions.
Non-Coding RNAs (ncRNAs): Some non-coding RNAs, such as small nucleolar RNAs (snoRNAs), play roles in rRNA transcription, processing, and modification. Their correct functioning is vital for the quality control of ribosome biogenesis.
Quality Control Mechanisms: Mechanisms that ensure the fidelity of rRNA transcription are part of the error check during ribosome biogenesis. These quality control mechanisms help detect and correct errors in the transcription process.
Nucleolar Proteins: Nucleolar proteins, including NOB1, XRN2, and other surveillance factors, participate in monitoring and maintaining the correctness of rRNA transcription and processing.
Ribosome Assembly Factors: Factors involved in ribosome assembly are often interconnected with rRNA transcription and processing. They ensure that the rRNA molecules are correctly processed and assembled into functional ribosomes.
Hormonal Regulation: Hormones can influence the rate of rRNA transcription in response to physiological changes and cellular demands. These hormonal signals are part of the regulatory network controlling ribosome production.

The regulation of rRNA transcription is a highly coordinated process that involves numerous players and mechanisms. It serves as a critical checkpoint to ensure that ribosomes are produced correctly and in the appropriate quantities to support cellular functions. This regulation is essential for maintaining the accuracy and efficiency of protein synthesis.

2. Error Detection during Translation

Mechanisms that identify mistakes or issues during the actual protein synthesis process.

Prokaryotic-Exclusive Mechanisms

Trans-Translation Mechanism 

A specialized rescue pathway exclusive to many prokaryotes, this mechanism addresses the stalling of ribosomes on defective mRNAs. Through the joint action of a unique RNA molecule known as transfer-messenger RNA (tmRNA) and its binding protein partner, SmpB, it releases the stalled ribosome, tags the incomplete polypeptide for proteolytic degradation, and targets the faulty mRNA for degradation. This dual action ensures the fidelity of translation and prevents the accumulation of potentially harmful truncated proteins in the cell. It's also noteworthy that trans-translation is not just a quality control mechanism but is harnessed in some genetic regulatory circuits to control gene expression. 13 The specialized rescue pathway exclusive to many prokaryotes is known as "trans-translation." This mechanism addresses the stalling of ribosomes on defective mRNAs and plays a crucial role in maintaining the fidelity of translation. Trans-translation involves the joint action of two key components: transfer-messenger RNA (tmRNA) and its binding protein partner, SmpB. Together, they perform several critical functions:

Ribosome Rescue: When a ribosome stalls on a defective mRNA, tmRNA and SmpB enter the rescue process. They help release the stalled ribosome from the problematic mRNA, preventing it from being trapped.
Tagging of Incomplete Polypeptide: Simultaneously, tmRNA and SmpB tag the incomplete polypeptide being synthesized within the stalled ribosome. This tag marks the polypeptide for proteolytic degradation.
Degradation of Faulty mRNA: In addition to tagging the incomplete polypeptide, the trans-translation complex also targets the faulty mRNA for degradation. This step ensures that the defective mRNA is removed from the translation machinery.

By performing these actions, trans-translation effectively prevents the accumulation of potentially harmful truncated proteins in the cell and maintains the integrity of the translation process. Importantly, trans-translation is not only a quality control mechanism but is also harnessed in some genetic regulatory circuits to control gene expression in prokaryotic cells. This dual role highlights its significance in cellular processes beyond error correction in translation.

Ribosome Collision Resolution 

Ribosomes are dynamic entities, and during translation, they move along the mRNA template. On occasions where a leading ribosome stalls, trailing ribosomes can catch up, leading to a "collision". While such collisions can also be observed in eukaryotes, the specific mechanisms to address these collisions are unique in prokaryotes and are vital for maintaining translation fidelity and preventing the formation of faulty proteins. Ribosome collision resolution is a critical process that ensures translation fidelity and prevents the formation of faulty proteins in prokaryotic cells. When multiple ribosomes are translating the same mRNA simultaneously, collisions between these ribosomes can occur. While similar collisions can also be observed in eukaryotic cells, the mechanisms to address these collisions are unique to prokaryotes.

In prokaryotic cells, ribosome collision resolution mechanisms are in place to manage these situations. The exact details of these mechanisms may vary, but the primary goals are to:

Prevent Stalling: The leading ribosome that stalls during a collision can disrupt translation and potentially lead to the formation of incomplete or faulty proteins. Collision resolution mechanisms aim to prevent this stalling.
Maintain Translation Fidelity: It's essential to maintain the accuracy of translation during and after a collision to ensure that the correct protein is synthesized.

While specific players or proteins involved in ribosome collision resolution may vary, some common features of these mechanisms include:

Ribosome Backtracking: In some cases, the leading ribosome may backtrack along the mRNA to allow the trailing ribosome to continue translation. This process can free the stalled ribosome.
Ribosome Recycling: In cases where the leading ribosome cannot resolve the collision by backtracking, ribosome recycling mechanisms may be employed. This involves disassembling the stalled ribosome and releasing the mRNA so that it can be retranslated.

These mechanisms, unique to prokaryotic cells, play a crucial role in maintaining translation fidelity and ensuring that functional proteins are produced, even in situations where ribosome collisions occur during translation.

Peptidyl Transferase Center Surveillance: The peptidyl transferase center is the ribosomal site where the nascent polypeptide chain is elongated by the addition of new amino acids. In prokaryotes, surveillance mechanisms at this center ensure the accurate and efficient formation of peptide bonds between incoming amino acids. Any deviations or abnormalities in this process can lead to the production of erroneous proteins, so this quality control step is crucial for maintaining cellular function and integrity.

Eukaryotic-Exclusive Mechanisms

Quality control mechanisms specific to eukaryotes play a vital role in maintaining the fidelity of protein synthesis. Given the intricacy of eukaryotic gene structure and regulation, these mechanisms serve as critical checkpoints to ensure correct mRNA processing and translation.

Exon Junction Complex (EJC) and Nonsense-Mediated Decay (NMD): In eukaryotes, genes are often interrupted by non-coding sequences called introns. Once transcribed, these introns are spliced out, and the remaining exons are joined together to form a mature mRNA. The splicing process leaves behind a molecular marker at exon-exon junctions, known as the Exon Junction Complex (EJC). The EJC serves multiple functions, one of which is the surveillance of mRNA for premature stop codons. When a translating ribosome encounters a premature stop codon before reaching and dislodging an EJC, this is an indication of a potentially faulty mRNA. In response, a quality control mechanism called Nonsense-Mediated Decay (NMD) is activated. NMD rapidly degrades these aberrant mRNAs, preventing the synthesis of truncated, and potentially harmful, proteins in the cell.
mRNA Surveillance and Exon Junction Complexes (EJCs): The EJC is not solely involved in detecting premature termination codons; it plays broader roles in mRNA surveillance. By marking exon-exon junctions, EJCs serve as reference points during translation, ensuring that ribosomes are correctly interpreting the coding sequence. EJCs also influence mRNA localization, transport out of the nucleus, and even the efficiency of translation itself. Their central role in coordinating these processes ensures that only properly spliced and processed mRNAs are translated, maintaining the fidelity and efficiency of protein synthesis in eukaryotic cells.

Together, these eukaryotic-exclusive mechanisms highlight the intricate layers of checks and balances that cater to the complexities of eukaryotic gene expression. They underscore the importance of precise coordination between mRNA processing and translation, ensuring that the vast array of eukaryotic proteins is synthesized accurately and efficiently.

Error Detection during Translation Extant in Both

The fidelity and efficiency of protein synthesis is a paramount objective shared by all cells, regardless of their complexity. Both prokaryotic and eukaryotic cells maintain a suite of mechanisms to safeguard the translation process, emphasizing the importance of accurate protein synthesis for cellular function and viability.

Chiral checkpoints during protein biosynthesis

"Chiral" is a term derived from the Greek word "χείρ" (cheir) which means "hand." In chemistry, chirality refers to the property of a molecule or an object that cannot be superimposed on its mirror image, just like how our left and right hands are mirror images of each other but are not superimposable. Molecules or objects that have this non-superimposable mirror image property are called "chiral." In the context of molecules, chirality often arises due to the presence of an asymmetric carbon atom, also known as a chiral center or stereocenter. This carbon is typically bonded to four different atoms or groups. The two non-superimposable mirror-image forms of a chiral molecule are called enantiomers. For instance, many biological molecules, such as amino acids and sugars, are chiral. In living organisms, usually, only one of the enantiomers is utilized. For example, in proteins, only L-amino acids (left-handed) are used, while D-sugars (right-handed) are used in nucleic acids. The chirality of a molecule can significantly influence its chemical behavior and interactions, especially in biological systems.

Chiral discrimination is pivotal and life-essential for several fundamental reasons:

Structural Integrity of Proteins: The three-dimensional structure of proteins is determined by the sequence and chirality of the amino acids they comprise. A switch from L- to D-amino acids would result in a drastically different three-dimensional conformation. The unique shapes and folds of proteins, guided by the specific sequence and chirality of amino acids, allow them to perform their specific functions.
Biochemical Specificity: Enzymes, which are a subset of proteins, act as biological catalysts and often recognize their substrates with high specificity. The "lock and key" model, where the enzyme (lock) perfectly fits with its substrate (key), is heavily dependent on the unique chiral shape of both the enzyme and substrate. The wrong chirality could prevent this interaction, making the enzyme non-functional.
Signal Transduction: Many signaling molecules are chiral, and their interactions with proteins (like receptors) are chirality-dependent. Incorrect chirality can hinder proper signal transmission or even result in erroneous signaling.
Genetic Code Consistency: The genetic code is standardized to correspond to L-amino acids. If D-amino acids were incorporated into proteins, it would represent a deviation from the genetic blueprint, potentially leading to non-functional or harmful proteins.

Life on Earth has standardized on L-amino acids (and D-sugars for nucleic acids). Switching chirality would be incompatible with the vast majority of life processes. D-amino acids are not typically used in eukaryotic cells for protein synthesis. If they were to be incorporated, they could be recognized as foreign or even toxic, potentially eliciting an immune response or leading to cellular dysfunction.  The ribosome plays a pivotal role in scrutinizing the handedness or chirality of the amino acids it incorporates into proteins. Given that only L-amino acids are utilized in biological protein synthesis, the ribosome acts as a "chiral checkpoint," rejecting D-amino acids and ensuring that only L-isomers are integrated. Chiral checkpoints during protein biosynthesis are essential mechanisms that ensure the correct incorporation of amino acids into proteins. In biological systems, only L-amino acids are utilized for protein synthesis, and the ribosome serves as a critical "chiral checkpoint" to enforce this specificity. This quality control mechanism prevents the incorporation of D-amino acids, which are not biologically relevant for protein synthesis, and ensures that only L-isomers are integrated into growing polypeptide chains. The ribosome achieves this chiral discrimination by recognizing and selectively binding to L-amino acids while rejecting D-amino acids. This selective binding process is crucial for maintaining the structural and functional integrity of proteins in living organisms. While the ribosome's role as a chiral checkpoint is well-established in biology, the exact molecular details of how it discriminates between L- and D-amino acids may vary among different organisms. Nevertheless, this mechanism is fundamental for the fidelity of protein synthesis, ensuring that the proteins produced in cells are composed of the correct building blocks with the appropriate chirality.

Ribosomal A-site: This is the active site of the ribosome where aminoacyl-tRNAs are accepted. The unique structural configuration of the A-site primarily allows binding and acceptance of L-aminoacyl-tRNA, while excluding D-aminoacyl-tRNA.
Aminoacyl-tRNA Synthetases: These enzymes are responsible for attaching amino acids to their corresponding tRNA molecules. They have high specificity for L-amino acids, ensuring that tRNAs are charged with the correct isomer.
Elongation Factor Tu (EF-Tu) in Bacteria or Elongation Factor 1A (EF-1A) in Eukaryotes: These factors deliver aminoacyl-tRNAs to the ribosome. They help ensure that only properly charged tRNAs (with L-amino acids) are presented to the ribosome.
Peptidyl Transferase Center (PTC): Located in the large subunit of the ribosome, the PTC facilitates peptide bond formation. Its structure is geared to preferentially accommodate L-amino acids, contributing to chiral discrimination.
Ribosomal RNA (rRNA): Forms the core of the ribosome and plays a role in catalyzing peptide bond formation. Specific nucleotides in the rRNA can interact with the ester linkage of L-aminoacyl-tRNA, ensuring the proper orientation and acceptance of L-amino acids.
tRNA Molecules: The L-shape of tRNA molecules and their specific anticodon loops ensure the correct reading of mRNA codons and, by extension, the delivery of the appropriate L-amino acid to the growing polypeptide chain.
Post-Translational Modification Enzymes: While not directly part of the ribosomal machinery, some enzymes can modify D-amino acids in specific proteins after synthesis. However, these are exceptions and not the rule in general protein synthesis.
Quality Control Factors: Some factors can be recognized when a ribosome stalls due to anomalies, including if a D-amino acid were to somehow be incorporated. They can trigger mechanisms like ribosome recycling or mRNA decay in response.

These components work in tandem to ensure the fidelity of protein synthesis, especially concerning the chirality of the amino acids being incorporated. The exact nuances of their roles and interactions might differ across organisms, but their collective function ensures the robustness and accuracy of the protein synthesis machinery. The orchestration of chiral discrimination during protein synthesis is an intricate and highly coordinated process involving multiple molecular players as outlined. The interdependence and complexity of these mechanisms raise questions about their initial creation and emergence.  Each component in the ribosomal machinery and its auxiliary factors has a specific role, and their functions are interconnected. For instance, aminoacyl-tRNA synthetases charge tRNAs with the appropriate L-amino acid, and then EF-Tu or EF-1A ensures that only these properly charged tRNAs are delivered to the ribosome. This sequential and cooperative functioning suggests that the individual components are not just independently operating units but are parts of an integrated system. The emergence of such a complex integrated system poses a timing conundrum. The components not only need to exist simultaneously but also need to function correctly right from the onset. A ribosome that does not discriminate chirality or an aminoacyl-tRNA synthetase that charges tRNAs indiscriminately would produce non-functional proteins, jeopardizing cell viability. The ribosome and the associated protein synthesis machinery are central to cellular function and life. Without precise chiral discrimination, cells would produce misfolded or non-functional proteins, leading to cellular chaos. Given that this machinery is essential for life, it presents a "chicken or the egg" dilemma: which component came first, and how could it function without the others? The high specificity and precision of this system are striking. Mistakes in chirality could have dire consequences, and the machinery has multiple checkpoints to ensure accuracy. Such precision implies that random or unguided processes would have had a minimal margin of error to produce a functional system. Considering the above points,  the emergence of such a system through unguided means poses significant is warranted to cause significant skepticism. The synchrony, specificity, and complexity of the chiral discrimination machinery indicate the requirement of foresight and capability of coordination that is difficult to reconcile with step-by-step, unguided processes, especially given the essential nature of the machinery for life. Thus, the origin of foundational and interdependent systems, such as the ribosomal machinery and its chiral discrimination, points overwhelmingly to design. The notion here is that the presence of such a coordinated and complex system, foundational for life, is plausibly explained as a designed setup rather than by an unguided, stepwise emergence by nonintelligent means.



mRNA translation regulation via tRNA modifications

Across both domains of life, tRNAs undergo a myriad of post-transcriptional modifications. With nearly 100 known epigenetic tRNA modifications, these chemical alterations play pivotal roles in modulating mRNA translation, affecting translational speed, accuracy, and overall protein output. mRNA translation regulation via tRNA modifications is a critical process that occurs in both prokaryotic and eukaryotic cells. Post-transcriptional modifications of tRNA molecules are widespread, with nearly 100 known epigenetic tRNA modifications. These chemical alterations are essential for fine-tuning and regulating mRNA translation, ultimately influencing translational speed, accuracy, and the overall output of proteins. The diversity of tRNA modifications allows cells to control various aspects of translation. These modifications can impact codon-anticodon interactions, the fidelity of translation, and the efficiency of ribosome movement along the mRNA template. As a result, tRNA modifications contribute to the precise regulation of protein synthesis, ensuring that the correct proteins are produced under specific cellular conditions. The intricate network of tRNA modifications underscores the complexity of mRNA translation regulation. Cells have evolved these mechanisms to adapt to various environmental cues and cellular requirements, allowing them to modulate protein synthesis with precision and efficiency. Whether in prokaryotic or eukaryotic cells, the role of tRNA modifications in mRNA translation regulation is a fundamental aspect of cellular biology.

Proofreading and editing processes

Central to the fidelity of protein synthesis is the ribosome's ability to ensure the faithful translation of genetic information from mRNA into protein. Through a combination of proofreading and editing steps, cells ensure that errors in amino acid incorporation are kept to a minimum. Proofreading and editing processes are crucial for maintaining the fidelity of protein synthesis in both prokaryotic and eukaryotic cells. These processes help ensure that genetic information encoded in mRNA is accurately translated into proteins, minimizing errors in amino acid incorporation. The ribosome, which serves as the central machinery for translation, plays a key role in these proofreading and editing mechanisms. In prokaryotic and eukaryotic cells, several mechanisms contribute to proofreading and editing during translation:

Aminoacyl-tRNA Synthetase Proofreading: Aminoacyl-tRNA synthetases are enzymes responsible for attaching amino acids to their corresponding tRNA molecules. These enzymes have proofreading capabilities to ensure that the correct amino acid is selected and attached to the tRNA. If an incorrect amino acid is attached, the enzyme can hydrolyze the incorrect aminoacyl-tRNA bond.
Ribosomal Proofreading: During translation, the ribosome monitors the accuracy of codon-anticodon interactions. If a mismatch occurs, the ribosome can stall and undergo a conformational change, allowing for the release of the incorrect aminoacyl-tRNA. This process, known as "kinetic proofreading," enhances the accuracy of translation.
tRNA Proofreading: Some tRNA molecules have editing sites that can hydrolyze mischarged amino acids, correcting any errors in amino acid selection.
Peptidyl Transferase Center (PTC) Proofreading: The PTC of the ribosome plays a role in monitoring the formation of peptide bonds between amino acids. It can detect and correct errors in peptide bond formation.
Ribosome-Associated Factors: Various ribosome-associated factors, such as EF-G in prokaryotes and eEF2 in eukaryotes, participate in proofreading mechanisms by facilitating the translocation of tRNA molecules and ensuring the fidelity of translation.

These proofreading and editing processes collectively contribute to the high accuracy of protein synthesis, ensuring that the final protein product accurately reflects the genetic information encoded in the mRNA. By minimizing errors in amino acid incorporation, cells can produce functional and correctly folded proteins essential for various cellular processes.

Chiral Discrimination by Ribosome

Reflecting the universal preference for L-amino acids in protein synthesis, ribosomes across all life forms have mechanisms to exclusively recognize and incorporate the L-isomer of amino acids, ensuring chiral purity in synthesized proteins. Chiral discrimination by ribosomes is a universal mechanism that ensures the incorporation of L-amino acids exclusively during protein synthesis, maintaining the chiral purity of synthesized proteins across all life forms. This mechanism reflects the preference for L-amino acids in biological systems and contributes to the fidelity and functionality of proteins in cells.

No-Go Decay

No-Go Decay (NGD): When ribosomes stall during translation, the No-Go Decay pathway is activated. This conserved mechanism recognizes the stalled ribosomes and leads to the endonucleolytic cleavage of the problematic mRNA. Key players in this mechanism include Pelota (Dom34 in yeast) and Hbs1, which target and bind to the stalled ribosomes. The downstream mRNA fragment is degraded by the 5'-3' exoribonuclease Xrn1, while the upstream fragment is degraded by the Ski complex and the Exosome in a 3'-5' direction.
Ribosome Recycling: After the problematic translation event, such as during NGD, ribosomal subunits need to be recycled for new rounds of translation. Rli1 (or ABCE1 in mammals) is an ATPase that assists in dissociating the ribosomal subunits, preparing them for another round of protein synthesis.
Ubiquitin-Proteasome Degradation: Incomplete or aberrant polypeptides resulting from stalled translation can be potentially harmful. These polypeptides are tagged for degradation using ubiquitin molecules by E3 ubiquitin ligases like Ltn1 (Listerin in mammals). Once ubiquitinated, these polypeptides are directed to the proteasome, an intricate protein complex, where they are degraded into amino acids.
Exosome-Mediated Decay: The Exosome is a multi-protein complex responsible for 3'-5' degradation of RNA. In the context of No-Go Decay, the Exosome plays a vital role in degrading the upstream mRNA fragments that result from the endonucleolytic cleavage at the stalled ribosome site.
Endonucleolytic Cleavage: One of the primary responses to ribosome stalling is the cleavage of mRNA in the A-site of the stalled ribosome. This cleavage results in the generation of upstream and downstream mRNA fragments, which are then targeted for degradation by various cellular machinery to prevent the accumulation of aberrant mRNAs.

Non-Functional rRNA Decay

In both prokaryotic and eukaryotic cells, the integrity of ribosomal RNAs (rRNAs) is essential for the accurate function of ribosomes in protein synthesis. When rRNAs are faulty or damaged, they can hinder the ribosome's role. The Non-Functional rRNA Decay (NRD) pathway comes into play to address this issue. This surveillance mechanism detects and degrades aberrant rRNAs, ensuring that only ribosomes with intact and functional rRNAs participate in translation. By doing so, the NRD pathway preserves the fidelity and efficiency of protein synthesis, safeguarding cellular functions.

Trm112: In yeast, Trm112 acts as a hub protein that associates with several methyltransferases, playing a role in 18S rRNA maturation. Mutations in associated methyltransferases or in Trm112 itself can lead to rRNA that's targeted by NRD.
Rrp6 and the Nuclear Exosome: In eukaryotes, aberrant pre-rRNAs and unassembled rRNA intermediates in the nucleus are targeted by the nuclear exosome, a complex with 3'-5' exoribonucleolytic activity. Rrp6 is one of the nuclear-specific catalytic subunits of this complex, and it is particularly essential for degrading erroneous rRNAs.
PARN: This 3'-5' exonuclease in humans is implicated in degrading the 3' external transcribed spacer (ETS) of pre-rRNA, acting as a surveillance mechanism to ensure proper rRNA maturation.
Mtr4: This RNA helicase is associated with the nuclear exosome and plays a role in guiding aberrant rRNAs and other faulty RNAs to the exosome for degradation.
Rat1 and Xrn1: These 5'-3' exonucleases in yeast are responsible for the degradation of the 5' ETS of pre-rRNA, ensuring the removal of improperly processed rRNA.
Nol12: A nuclear protein in humans that, when mutated, can lead to the accumulation of aberrant pre-rRNAs, suggesting its role in proper rRNA processing and potentially in NRD.
RACK1: In eukaryotic ribosomes, RACK1 is a component of the 40S subunit. It has been suggested to play a role in recognizing and targeting defective 18S rRNAs for degradation, although its exact mechanism in NRD is still under investigation.
HEAT Repeat Proteins: These are suggested to be involved in recognizing aberrant rRNAs and targeting them for degradation, ensuring ribosomal quality control.

This list provides a snapshot of some of the key players in the NRD pathway, although the complete picture is more complex and may vary between organisms. It's worth noting that while the NRD pathway is essential for cellular health, much about its mechanisms and associated factors remains an active area of research.

mRNA Surveillance and Decay

To ensure fidelity and efficiency in gene expression, cells have several mRNA surveillance pathways that identify and degrade aberrant mRNAs or those that are no longer needed. Here are the primary surveillance mechanisms and the key players involved:

Nonsense-Mediated Decay (NMD): This pathway identifies mRNAs that contain premature stop codons to prevent the production of truncated and potentially harmful proteins.

Upf1, Upf2, and Upf3: Core components of the NMD pathway. Upf1 is an RNA helicase, and when bound to mRNA, can initiate decay if it interacts with the exon-junction complex (EJC) through Upf2 and Upf3.
SMG1-7 proteins: In mammals, these assist Upf1 in promoting decay, with SMG1 having a role in Upf1 phosphorylation.
Non-Stop Decay (NSD): Targets mRNAs that lack stop codons, preventing the ribosome from releasing and potentially leading to problematic polypeptides.
Ski7: In yeast, this protein recognizes ribosomes stuck on non-stop mRNAs and promotes their degradation.
Pelota/Dom34 and Hbs1: These recognize and bind to the stalled ribosomes on non-stop mRNAs, leading to their release.
5' to 3' mRNA Decay: Targets mRNAs from the 5' end.
Dcp1/Dcp2 complex: Initiates decapping of the mRNA.
Xrn1: A 5'-3' exoribonuclease that degrades the mRNA once the cap is removed.
3' to 5' mRNA Decay: Targets mRNAs from the 3' end.
The Exosome: Multi-protein complex responsible for the 3'-5' degradation of RNA.
Decapping Mediated Decay: Targets mRNAs by removing the protective 5' cap structure.
Edc1-4 and Lsm1-7: Proteins that promote decapping and subsequent degradation of the mRNA body.
mRNA Quality Control at the Ribosome: Identifies issues that arise during active translation.
ZNF598: Recognizes ribosomes that stall at the site of a collision and subsequently triggers mRNA decay pathways.

This list provides a snapshot of some of the key players in various mRNA surveillance pathways. Each pathway ensures that only properly processed and functional mRNAs are translated, while problematic ones are quickly and efficiently removed.

Polysome Surveillance: Polysomes, clusters of ribosomes translating a single mRNA simultaneously, are subject to surveillance. This mechanism ensures that polysomes function efficiently and are devoid of defects that might hinder translation.
Translation Fidelity Checkpoints: To maintain the accuracy of protein synthesis, cells employ various checkpoints during translation. These systems verify the correct decoding of mRNA sequences and the appropriate incorporation of amino acids into the growing polypeptide chain.
Ribosome Function Monitoring: The accurate matching of tRNAs to mRNA codons is a cornerstone of translation fidelity. Ribosomes possess intrinsic mechanisms to monitor this matching process, ensuring that the correct amino acid-tRNA conjugates are selected in response to the codons of the mRNA template.

3. Error Correction during Translation

Once an error is detected, these mechanisms work to correct it.

Prokaryotic-Exclusive Mechanisms

Ribosome Assembly in Response to Temperature Changes

The ribosome assembly process in bacteria is indeed sensitive to temperature fluctuations, and this sensitivity plays a crucial role in the bacteria's ability to adapt to varying environmental conditions. Several molecular players and factors are involved in this temperature-responsive mechanism:

rRNA (Ribosomal RNA): rRNA transcription and its correct folding are critical for ribosome assembly. Temperature changes can affect the secondary and tertiary structures of rRNA, which can, in turn, affect ribosome biogenesis.
Ribosomal Proteins: The binding of ribosomal proteins to rRNA is temperature-dependent. Some proteins bind more efficiently at certain temperatures, ensuring the structural stability of the ribosome.
RNA Chaperones: These are proteins like Hfq and ProQ, which help in the correct folding of rRNA and other RNAs at varying temperatures.
RNA Helicases: Proteins such as RhlE, RhlB, and DeaD help in the remodeling of RNA structures, especially under temperature stress. They can resolve misfolded RNA structures, facilitating the correct assembly of ribosomal components.
RNA Thermometers: These are specific RNA structures present in some mRNA molecules, and they can undergo conformational changes in response to temperature variations. By doing so, they regulate the translation of associated genes, including those involved in ribosome assembly and stress responses.
Small Nucleolar RNAs (snoRNAs): While they are more prominent in eukaryotes, some bacteria also possess snoRNAs that assist in rRNA processing, modification, and ribosome assembly.
Chaperone Proteins: Proteins like DnaK, DnaJ, and GroEL/GroES are involved in the folding and stabilization of other proteins, including ribosomal proteins, especially under temperature stress.
Stringent Response: This is a stress response in bacteria that is activated under various conditions, including temperature stress. The alarmone molecules (ppGpp and pppGpp) produced during the stringent response can regulate ribosomal RNA synthesis, ensuring that ribosome production is tuned according to the growth conditions.

Ribosome Modifying Enzymes 

Ribosome-modifying enzymes play crucial roles in the post-transcriptional and post-translational modifications of ribosomal components. While some of these modifications are essential for the ribosome's general function, others may help adapt the ribosome's activity in response to environmental changes, including temperature fluctuations. Here are some key ribosome-modifying enzymes:

Pseudouridine Synthases: These enzymes convert uridine residues in rRNA to pseudouridine. Examples include RluA, RluB, RluC, RluD, RluE, and RluF in bacteria.
Methyltransferases: These enzymes add methyl groups to specific bases or the ribose backbone of rRNA. Examples are RsmA, RsmB, RsmC, RsmD, RsmE, RsmF, and RsmG in bacteria.
Dimethyltransferases: Enzymes like KsgA add two methyl groups to specific adenine residues in rRNA.
RNA Deaminases: These enzymes deaminate specific adenosine residues to inosine in rRNA.
Ribosomal Protein Acetyltransferases: These enzymes modify specific ribosomal proteins by adding acetyl groups. An example in bacteria is RimI, which acetylates ribosomal protein S18.
Ribosomal Protein Methyltransferases: Enzymes like PrmA trimethylate lysine residues on ribosomal proteins.
Ribosomal Protein Phosphotransferases: These enzymes, although more common in eukaryotes, phosphorylate ribosomal proteins, potentially altering their activity and interactions.
Ribosomal Protein Ubiquitin Ligases: More commonly found in eukaryotes, these enzymes attach ubiquitin to ribosomal proteins, signaling them for degradation or altering their function.
Ribonucleases: Specific RNases may trim or process rRNA molecules to ensure their proper maturation and function.

It's worth noting that while these enzymes contribute to the overall modification landscape of the ribosome, not all may be directly implicated in temperature-responsive adaptations. However, the post-transcriptional and post-translational modifications they confer can influence ribosome structure and function, potentially aiding the ribosome in functioning optimally under varying temperature conditions.

Degradation and Turnover Machinery

Lon Protease: A major ATP-dependent protease in bacteria, targeting misfolded proteins for degradation, including malfunctioning ribosomal proteins.
ClpXP Protease: Recognizes and degrades misfolded or unneeded proteins in bacteria, potentially including ribosomal proteins.
FtsH (HflB) Protease: An integral membrane protease in bacteria that degrades certain misfolded membrane proteins and ribosomal proteins.
La Protease: This protease, present in some bacteria, targets and degrades specific pre-tRNA species.
RNase R: A 3'-5' exoribonuclease involved in rRNA decay.
RNase II: Another 3'-5' exoribonuclease in bacteria that is involved in the degradation of many RNA species, potentially including faulty rRNAs.
Pnp (Polynucleotide Phosphorylase): Functions both as a polymerase and a 3'-5' exoribonuclease, involved in the decay of several RNA species.
RNase E: Part of the RNA degradosome in bacteria, playing a significant role in rRNA processing and mRNA decay.
RNA Degradosome: A multi-protein complex in bacteria like *Escherichia coli*, involved in the coordinated degradation of various RNA species.
Endonuclease YbeY: An endoribonuclease in bacteria involved in the maturation and quality control of the 70S ribosome.
Dom34 (Pelota) and Hbs1: In eukaryotes, they recognize and target stalled ribosomes, leading to ribosome recycling.
Exosome Complex: Present in both eukaryotes and archaea, this multi-protein complex is responsible for 3'-5' degradation of various RNA species.

These molecular players operate in coordination to ensure that ribosome assembly is appropriately modulated in response to temperature changes. The precise roles and interactions of these components can vary among different bacterial species and strains, reflecting the diverse strategies bacteria employ to thrive in various environments.

rRNA Methyltransferase Counteraction

Specific methyltransferases in prokaryotes add methyl groups to rRNA. This modification can provide resistance against certain antibiotics by preventing them from binding to the ribosome. This is especially crucial for bacteria in dynamic environments where antibiotic exposure might be sporadic.

Erm Methyltransferases: These enzymes methylate adenine residues in 23S rRNA. Methylation at this position confers resistance to macrolide, lincosamide, and streptogramin B antibiotics. Examples include ErmA, ErmB, and ErmC.
Cfr Methyltransferase: This enzyme methylates adenine residues in 23S rRNA, conferring resistance to multiple classes of antibiotics including phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A.
RsmG Methyltransferase: Methylates guanine at position 966 of 16S rRNA in certain bacteria, impacting the binding of aminoglycoside antibiotics.
RsmA and KsgA Methyltransferases: They methylate two adjacent adenines (A1518 and A1519 in E. coli numbering) in 16S rRNA. KsgA methylation can decrease aminoglycoside antibiotic sensitivity.
Rmt Methyltransferases: These are 16S rRNA methyltransferases that confer high-level resistance to aminoglycosides. Examples include RmtA, RmtB, RmtC, and RmtD.
NpmA Methyltransferase: Specifically methylates the 16S rRNA, leading to resistance against aminoglycosides.
TlyA Methyltransferase: This enzyme is unique in that it can methylate both 23S and 16S rRNA, leading to resistance against certain drugs, including capreomycin.
RNA Polymerase: While not a methyltransferase, mutations in RNA polymerase can also confer resistance to rifamycin antibiotics.
Efflux Pumps: Again, not methyltransferases, but they are proteins that actively transport antibiotics out of the bacterial cell, lowering the intracellular concentration of the drug. Examples include the AcrAB-TolC system in E. coli.
Antibiotic Modifying Enzymes: These enzymes can directly modify antibiotics, rendering them ineffective. Examples are beta-lactamases that hydrolyze the beta-lactam ring of penicillin-like antibiotics.

Feedback-Controlled Ribosome Synthesis

In bacteria, the synthesis of ribosomal proteins is intricately balanced to ensure that the right amount of proteins are produced for ribosome assembly. One of the ways bacteria achieve this is through feedback control mechanisms. Specifically, when an excess of a certain ribosomal protein is present in the cell and hasn't been incorporated into a ribosome, that protein can bind to its own mRNA. By binding to the mRNA, the ribosomal protein often prevents the translation of its own mRNA, thereby halting its own synthesis. This feedback inhibition ensures that resources are not wasted on producing excess ribosomal proteins and maintains a balance between the various ribosomal components. Key Mechanisms in Feedback-Controlled Ribosome Synthesis:

mRNA Binding: Many ribosomal proteins can specifically bind to their own mRNAs. This binding usually occurs in the regions of the mRNA that correspond to the protein's coding sequence. By binding, these proteins often occlude the ribosome-binding site on the mRNA, preventing further translation.
Anti-terminator Activity: In some instances, ribosomal proteins bind to their mRNA in a way that prevents the formation of a transcription terminator hairpin structure. This can lead to the continuation of transcription beyond the ribosomal protein genes, which might include downstream genes necessary for rRNA synthesis or other functions.
Translational Repressors: Some ribosomal proteins act as translational repressors when they bind to their own mRNA, blocking the initiation of translation.
Ribosomal Assembly Checkpoints: As ribosomal proteins and rRNAs come together to form mature ribosomal subunits, there's a preferential order and assembly pathway. If certain proteins are in excess, they may not find an available assembly slot, triggering the feedback mechanism.
Ribosome Rescue and Quality Control: If errors occur during ribosome assembly or if damaged ribosomal components are detected, there are quality control mechanisms in place, like tmRNA, which helps release stalled ribosomes, and various proteases and nucleases, which degrade aberrant ribosomal components.

This feedback-regulated system is a sophisticated way bacteria optimize resource utilization and ensure the efficient production and assembly of their ribosomal machinery. It also underscores the precision and adaptability of bacterial regulatory systems in response to changing intracellular conditions.

Modulation of rRNA Promoters

the regulation of rRNA transcription in prokaryotes is a complex process that's crucial for adapting to various environmental conditions and cellular demands. Modulating the activity of rRNA promoters enables cells to efficiently adjust ribosome production, optimizing protein synthesis capacity based on need. Mechanisms and Players in Modulation of rRNA Promoters:

Sigma Factors: In bacteria, different sigma factors can direct RNA polymerase to specific sets of promoters. The primary sigma factor (like Sigma-70 in E. coli) recognizes most rRNA promoters under normal growth conditions. However, under specific conditions, alternative sigma factors can be used to recognize different promoters, modulating rRNA transcription.
Stringent Response: This is a regulatory pathway activated under nutrient limitation or stress. One of its primary effects is the dramatic reduction in rRNA and tRNA synthesis. When amino acid levels drop, uncharged tRNAs activate the enzyme RelA to synthesize an alarmone molecule called (p)ppGpp. This molecule interacts with RNA polymerase, reducing its affinity for rRNA promoters and thus decreasing rRNA transcription.
Nus Factors: These are proteins like NusA and NusB that influence the elongation phase of transcription and can affect rRNA gene transcription.
Antitermination Mechanisms: Certain proteins can prevent the formation of terminator structures in the newly synthesized RNA, allowing RNA polymerase to continue transcription. In the context of rRNA, this ensures that the entire rRNA operon is transcribed efficiently.
DNA Supercoiling: The level of DNA supercoiling can influence promoter activity. Changes in supercoiling can be a response to environmental factors like temperature or osmolarity. Topoisomerases, enzymes that modulate DNA supercoiling, thus play an indirect role in rRNA promoter activity.
Small Molecules and Metabolites: Molecules like cyclic AMP (cAMP) can influence rRNA transcription. In some bacteria, when glucose levels are low, cAMP levels rise, which in turn affects the transcription of various genes, including potentially rRNA genes.
Transcription Factors and Repressors: Various protein factors can bind directly to rRNA promoters or nearby sequences to either enhance or repress transcription initiation. These can respond to different signals or conditions to modulate rRNA synthesis.

These diverse mechanisms, and their integration, allow bacteria to fine-tune ribosome production in response to their environment and metabolic status. Given that ribosome biogenesis is a resource-intensive process, this regulation is crucial for cellular efficiency and adaptability.

Ribosome Hibernation Promotion

Ribosome hibernation is a fascinating strategy that certain bacteria employ to survive in adverse environmental conditions. During this process, the bacterial ribosomes are converted into translationally inactive 100S complexes, which can be rapidly reactivated when conditions become favorable. Mechanisms and Players in Ribosome Hibernation:

Hibernation-promoting factor (HPF): Found in many bacteria, this protein binds to the 30S ribosomal subunit, stabilizing the dimerization of 70S ribosomes into inactive 100S complexes.
Ribosome modulation factor (RMF): Present in some bacteria like E. coli, RMF binds to the 50S subunit, facilitating the formation of the inactive 90S dimer. This can then further dimerize with the help of HPF to form the 100S complex.
YfiA (pY): Found in certain bacteria, YfiA inhibits translation by binding to the 30S subunit, preventing association with the 50S subunit.
Short hibernation-promoting factor (sHPF): In some Gram-negative bacteria, a shorter version of HPF has been identified. While its mechanism is similar to HPF, it's evolved differently and seems to be more associated with certain pathogenic bacteria.
RelA and (p)ppGpp: Although not directly involved in ribosome hibernation, the stringent response, mediated by RelA and the alarmone (p)ppGpp, can reduce the overall translation activity in the cell. This can indirectly promote ribosome hibernation as a part of the general response to stress.
Ribosome-Associated Quality Control (RQC): In some cases, the hibernation process might be linked with RQC. If a ribosome gets stalled during translation, specific factors can target it for disassembly or hibernation.

The process of ribosome hibernation, and its subsequent reactivation, is a remarkable example of bacterial adaptability. By temporarily putting their protein synthesis machinery on hold, bacteria can endure conditions that might be detrimental to active ribosomes. Once conditions improve, these hibernating ribosomes can be swiftly reactivated, allowing the bacteria to quickly resume growth and proliferation. This capability provides a distinct survival advantage in fluctuating environments.

Hibernation in the Macro World

In the animal kingdom, numerous creatures undergo hibernation, a state of inactivity and metabolic depression, to survive prolonged periods of extreme cold or scarcity of food. For instance, bears will lower their heart rate, decrease their metabolic rate, and recycle waste products during winter months. This isn't merely a passive process. It's an intricate physiological adaptation that involves a multitude of finely regulated processes: Before hibernation, animals accumulate fat reserves, which will serve as their primary energy source during hibernation. During hibernation, body temperature drops to save energy. Some hibernators can recycle waste products, such as urea, to prevent toxin accumulation. Even after months of inactivity, hibernating animals can wake up without significant muscle loss. On a much smaller scale, certain bacteria transition ribosomes into a dormant "hibernation" state using hibernation-promoting factors. This strategy conserves cellular resources and allows bacteria to swiftly resume translation when conditions become favorable again.

Convergent Mechanism - A Parallel in Design

What's truly intriguing is the convergence of this mechanism across vastly different scales and domains of life. While the molecular and physiological processes in ribosome hibernation and animal hibernation are distinct, the underlying principle is the same: both entail a strategic metabolic downturn to conserve energy and endure unfavorable conditions, followed by a rapid reactivation when conditions improve. Such parallel strategies, seen both at the macroscopic level in mammals and at the microscopic level in bacteria, can be seen as indicative of a design principle. In the realm of human-made designs, recurring solutions and patterns are employed to solve similar challenges, even in very different contexts. Why would this principle not apply to the natural world? The existence of hibernation in both macro and micro realms suggests a designer who implemented similar solutions across different scales and domains of life, demonstrating foresight, efficiency, and an elegant sense of continuity. Convergence in biological systems, especially when it serves a similar functional purpose across diverse organisms, can be perceived as more than mere coincidence. It can be viewed as evidence of a purposeful design, implemented by an entity with an understanding of both the vastness and intricacies of life.



Eukaryotic-Exclusive Mechanisms

Anti-Association Factors 

In eukaryotes, anti-association factors play a critical role in ensuring that ribosomal subunits, specifically the large and small subunits, do not associate prematurely in the absence of mRNA. This mechanism ensures that only translationally competent ribosomes, which have properly engaged with mRNA, initiate protein synthesis.

Anti-Association Factors: These factors are crucial in eukaryotes to prevent the premature association of ribosomal subunits, especially the large and small subunits, in the absence of mRNA. Their presence ensures that ribosomes only initiate protein synthesis when they are translationally competent and have correctly engaged with mRNA.
Ribosomal Subunits: These are the large and small components of the ribosome that come together to facilitate protein synthesis. Anti-association factors prevent their premature association without mRNA.
mRNA (Messenger RNA): mRNA provides the template for protein synthesis. It ensures that ribosomes, after proper engagement, initiate the translation process.
Translationally Competent Ribosomes: These are ribosomes that have properly engaged with mRNA and are ready to start the process of protein synthesis.

Error Correction during Translation Extant in Both

Ribosome-Associated Quality Control (RQC)

The RQC pathway plays a critical role in recognizing ribosomes that have stalled during translation, ensuring that the incomplete polypeptides produced by these ribosomes are targeted for degradation. This system acts as a safeguard against the accumulation of potentially harmful protein fragments inside the cell.

Recognition of Stalled Ribosomes:
Stalled ribosomes are identified when translation is hindered due to reasons such as mRNA truncation, rare codon clusters, or amino acid starvation.
Dom34/Hbs1 (eukaryotes) or tmRNA (prokaryotes): Systems that recognize and rescue stalled ribosomes.
       
Ribosome Splitting
Once a stalled ribosome is identified, the large and small subunits of the ribosome are dissociated to release the incomplete polypeptide.
Dom34/Hbs1 (eukaryotes): Assists in splitting the ribosome.
Rli1/ABCE1 (eukaryotes): ATP-binding cassette protein that plays a role in ribosome recycling.

Tagging of Incomplete Polypeptides:
Incomplete polypeptides are tagged to be recognized by the cellular degradation machinery.
Ltn1 (Listerin in eukaryotes): An E3 ubiquitin ligase that adds ubiquitin chains to the incomplete polypeptides, marking them for degradation.

Degradation of Incomplete Polypeptides:
After tagging, the incomplete polypeptides are targeted for degradation to prevent their accumulation.
Proteasome (eukaryotes): A large protein complex responsible for degrading ubiquitinated proteins.
ClpXP and Lon proteases (prokaryotes): Recognize and degrade tagged proteins.

mRNA Quality Control:
In parallel to the degradation of incomplete polypeptides, the mRNA that led to ribosome stalling is also targeted for degradation to prevent further stalled translation events.
No-Go Decay (NGD) (eukaryotes): Targets mRNAs that cause ribosomal stalling for degradation.
Xrn1 (eukaryotes): 5' to 3' exonuclease that degrades problematic mRNAs.
Ski complex (eukaryotes): Assists in the 3' to 5' degradation of problematic mRNAs.

Recycling of Ribosomal Components:
After the resolution of the stall, ribosomal components are recycled to participate in new rounds of translation.
Rli1/ABCE1 (eukaryotes): Facilitates the recycling of ribosomal subunits.

By having these mechanisms in place, cells ensure that translation remains accurate and efficient and that potential errors are swiftly corrected to maintain cellular homeostasis.

rRNA Mismatch Correction

rRNA Mismatch Correction:

The precision of rRNA sequences is vital for the proper functioning of ribosomes. Both prokaryotic and eukaryotic cells have devised intricate surveillance systems to detect and rectify mismatches within rRNA, ensuring the ribosomes maintain their optimal activity.

Recognition of Mismatches: The initial step involves identifying any aberrant bases within the rRNA that could potentially affect ribosome function.
snoRNAs (eukaryotes): Small nucleolar RNAs guide the modification of specific bases in rRNAs and play a role in mismatch detection.
rRNA methyltransferases (prokaryotes): Enzymes that modify rRNA bases, their activity can be hindered by mismatches, leading to recognition.

Mismatch Repair: Once detected, the mismatched bases are repaired to maintain the integrity of the rRNA sequence.
RNases (both cell types): Enzymes that can cleave the rRNA at the site of the mismatch.
RNA Polymerases (both cell types): Enzymes responsible for synthesizing new rRNA sequences to replace the mismatched sections.

Quality Control & Degradation: If the mismatch is irreparable, the affected rRNA molecule is marked for degradation to prevent its incorporation into a ribosome.
TRAMP complex (eukaryotes): Assists in marking the faulty rRNA for degradation by the exosome.
Exosome complex (eukaryotes): Degradation machinery for faulty rRNA.
PNPase (prokaryotes): Polynucleotide Phosphorylase, involved in the degradation of aberrant rRNA.

Surveillance & Recycling: The cell ensures that the components of the degraded rRNA are recycled and reused.
Exosome complex (eukaryotes): Apart from degradation, it also plays a role in recycling rRNA components.
Rrp44/Dis3 (eukaryotes): An exoribonuclease component of the exosome involved in the processing and recycling of rRNA components.

Through these mechanisms, cells maintain the fidelity of their rRNA sequences, ensuring the ribosomes function efficiently in protein synthesis.

Ribosomal RNA Pseudouridylation 

Pseudouridylation is a pivotal post-transcriptional modification that serves to enhance the stability and function of rRNA across both prokaryotic and eukaryotic domains. This alteration involves the isomerization of uridine to pseudouridine within the rRNA, lending structural rigidity and functional precision to the ribosome.

Mechanism of Pseudouridylation: The process is catalyzed by specific enzymes or guided by RNAs that identify target uridines for conversion.
Pseudouridine synthases (both cell types): Enzymes responsible for catalyzing the conversion of uridine to pseudouridine directly.
snoRNAs (eukaryotes): Small nucleolar RNAs guide the site-specific isomerization of uridine in eukaryotic rRNA. They form base-pairing interactions with target sites.
sRNA-guided mechanisms (prokaryotes): In bacteria, specific small RNAs can serve a similar role to snoRNAs in guiding the pseudouridylation process.

Functional Implications: Pseudouridylation offers both structural and functional advantages to the ribosome.
rRNA stabilization: The presence of pseudouridine introduces additional hydrogen bonding, enhancing the overall stability and integrity of rRNA structures.
Modulation of ribosome activity: Pseudouridylation can influence ribosomal decoding accuracy, translational fidelity, and responsiveness to cellular stress conditions.

Quality Control & Surveillance: Ensuring the correct positioning and number of pseudouridines is crucial for ribosome function.
rRNA modification monitoring (both cell types): Cells possess mechanisms to monitor and ensure the correct modification of rRNA, including pseudouridylation.
Exosome complex (eukaryotes): Targets improperly modified rRNA, including incorrectly pseudouridylated rRNA, for degradation.

Regulation & Dynamics: Pseudouridylation patterns can change in response to cellular conditions.
Environmental stress (both cell types): Can lead to alterations in rRNA pseudouridylation patterns, enabling the ribosome to adapt to changing cellular conditions.
Developmental stages (eukaryotes): Different pseudouridylation patterns might be observed during various developmental stages, indicating a potential regulatory role in gene expression.

Through the concerted action of enzymes, guide RNAs, and surveillance mechanisms, cells ensure the appropriate pseudouridylation of rRNA, thus optimizing ribosomal function and adaptability.

E-site Quality Control 

The E-site (exit site) of the ribosome is the final station for tRNAs during protein synthesis, ensuring the proper and efficient completion of translation. It's paramount that the tRNA's exit from this site is closely monitored and regulated, preventing any potential hitches in the overall translational process.

Function of the E-site: The E-site serves as the exit door for tRNAs that have already contributed their amino acid to the growing polypeptide chain.
Deacylated tRNAs: After donating their amino acid, tRNAs become deacylated and are positioned in the E-site, awaiting their exit from the ribosome.

Mechanisms overseeing the E-site: Various factors and processes ensure that tRNAs efficiently depart from the E-site, maintaining the rhythm and fidelity of translation.
Elongation factors (both cell types): Facilitate the progression of tRNAs through the ribosomal A, P, and E sites, ensuring a smooth and timely exit of deacylated tRNAs from the E-site.
Ribosomal rRNA and proteins: Interact with tRNAs and play a role in ensuring the correct positioning and release of tRNAs from the E-site.
Post-translational modifications: Some modifications on tRNAs can influence their interaction dynamics with the ribosome, impacting their release from the E-site.

E-site associated quality control mechanisms: Missteps in translation can lead to aberrant polypeptides; thus, mechanisms are in place to oversee the E-site function.
RQC (Ribosome-associated Quality Control) (eukaryotes): Detects stalled ribosomes and targets aberrant nascent polypeptides for degradation, ensuring that tRNAs exit the E-site as intended.
tmRNA system (prokaryotes): Rescues ribosomes stalled on mRNAs without stop codons, simultaneously ensuring proper E-site tRNA exit.

Regulation of E-site dynamics: The departure of tRNAs from the E-site can be modulated based on cellular conditions.
Cellular stress (both cell types): Under certain stress conditions, ribosomal stalling can occur, which might impact E-site dynamics. Cells have adaptive responses to deal with such scenarios, ensuring efficient tRNA exit from the E-site.
Ribosome biogenesis and modifications: Variations in ribosome composition or modifications can influence E-site dynamics, potentially modulating translation rates and fidelity.

Through a combination of ribosomal components, translational factors, and quality control mechanisms, cells ensure the accurate and efficient functioning of the E-site, pivotal for the completion of protein synthesis.

tRNA Aminoacylation Fidelity

Aminoacyl-tRNA synthetases in both eukaryotes and prokaryotes ensure that tRNAs are charged with their corresponding, cognate amino acids. This process is crucial for maintaining the fidelity of protein synthesis.

tRNA Aminoacylation Fidelity:

The accuracy of tRNA aminoacylation, where tRNAs are charged with their respective amino acids, is foundational for the fidelity of protein synthesis. Aminoacyl-tRNA synthetases play the pivotal role in ensuring this precision across both eukaryotic and prokaryotic cells.

Purpose of Aminoacylation:
Aminoacylation involves attaching the correct amino acid to its corresponding tRNA, ensuring that the genetic code is translated accurately into proteins.
Aminoacyl-tRNA: The product of this process, it's a tRNA molecule charged with its specific amino acid.

Aminoacyl-tRNA Synthetases:
These enzymes are responsible for the correct matching of amino acids to their respective tRNAs.
Recognition pockets: Each synthetase has specific binding sites that can recognize and bind to both a specific amino acid and its corresponding tRNA.
Activation sites: These sites are where the amino acid is activated by ATP, facilitating its attachment to tRNA.

Ensuring Aminoacylation Fidelity:
Mistakes in aminoacylation can lead to misfolded proteins; thus, multiple mechanisms ensure the process's accuracy.
Editing sites (for certain synthetases): These sites can remove incorrectly attached amino acids, serving as a correction mechanism.
Proofreading: Some aminoacyl-tRNA synthetases have an additional proofreading step to ensure the correct amino acid is attached.
tRNA modifications: Modifications on tRNAs can influence their recognition by synthetases, enhancing fidelity.

Consequences of Aminoacylation Errors:
Inaccurate aminoacylation can lead to misincorporation of amino acids in proteins, affecting their structure and function.
Misfolded proteins: These can be non-functional or even harmful to the cell.
Cellular stress responses: Cells can detect and respond to the presence of misfolded proteins, activating pathways to either refold or degrade them.

Regulation of Aminoacylation:
The efficiency and accuracy of tRNA aminoacylation can be modulated based on cellular needs and conditions.
Amino acid availability: The availability of amino acids can influence the rate and accuracy of tRNA charging.
Post-translational modifications: Some aminoacyl-tRNA synthetases can be modified after their synthesis, potentially influencing their activity and specificity.

Through the precise action of aminoacyl-tRNA synthetases and associated quality control mechanisms, cells ensure the correct translation of genetic information, producing functional proteins essential for cellular health and function.

Correct Positioning of tRNAs

The precise placement of tRNAs within the ribosome during translation is fundamental to the accurate synthesis of proteins. Both eukaryotic and prokaryotic cells employ intricate mechanisms to ensure that tRNAs are correctly oriented and positioned.

Three tRNA Binding Sites on the Ribosome: A-site (Aminoacyl site): The entry site for the aminoacyl-tRNA corresponding to the next codon on the mRNA.
P-site (Peptidyl site): Holds the tRNA with the growing peptide chain.
E-site (Exit site): Where deacylated tRNAs depart after donating their amino acid.

Ensuring Proper tRNA Positioning:
Codon-Anticodon Base Pairing: Ensures that the correct aminoacyl-tRNA enters the A-site based on the mRNA codon.
Ribosomal rRNA: Plays a role in stabilizing the tRNA positions and ensuring their proper orientation within the ribosomal sites.
EF-Tu (in prokaryotes) or eEF1A (in eukaryotes): Proteins that deliver the aminoacyl-tRNA to the A-site. They undergo conformational changes upon GTP hydrolysis to ensure correct tRNA placement.
Peptidyl transferase center: Located within the ribosome, it ensures the correct orientation of tRNAs for peptide bond formation between the amino acids.

Checking tRNA Positioning:
Ribosome conformational changes: The ribosome undergoes subtle shifts to accommodate and check the tRNAs, providing an additional layer of quality control.
Release factors: Recognize stop codons and ensure that the terminating tRNA, rather than aminoacyl-tRNA, is positioned in the A-site to terminate translation.

Consequences of Incorrect tRNA Positioning:
Frame-shifting: Incorrect positioning can lead to reading the mRNA in the wrong frame, resulting in the synthesis of aberrant proteins.
Premature termination or elongation: Mispositioned tRNAs can lead to the premature end of translation or the incorrect continuation of translation.
Cellular stress responses: Cells can detect aberrant proteins resulting from translation errors and activate pathways to either refold or degrade them.

Regulation and Adaptation:
Ribosome-associated factors: Some proteins associate with ribosomes to aid in tRNA positioning, especially under specific cellular conditions or stresses.
Post-transcriptional modifications: Modifications on tRNAs and rRNAs can influence their interaction and positioning within the ribosome.

Through these stringent mechanisms and checks, cells ensure the correct positioning of tRNAs, facilitating the accurate translation of genetic information into functional proteins.

Ribosome-Templated Protein Aggregation

Misfolded or aggregated proteins can pose a threat to cellular function and can potentially stall or impair ribosomes during translation. Both eukaryotic and prokaryotic cells have evolved intricate systems to address such protein aggregates, either attempting to refold them or marking them for degradation.

Reasons for Protein Aggregation on Ribosomes:
Translation errors: Incorrect amino acid incorporation can lead to misfolded proteins.
Oxidative stress: Damages amino acids, affecting protein folding.
Temperature changes: High temperatures can denature proteins.
Mutations: Altered protein sequences may not fold correctly.

Cellular Response to Aggregated Proteins:
Ribosome-associated protein quality control (RQC): Recognizes stalled ribosomes and recruits factors to refold or degrade the problematic protein.
Heat shock proteins (HSPs): Chaperones that aid in refolding misfolded proteins.
Proteasome (in eukaryotes): A complex responsible for degrading ubiquitinated proteins.
Lon and ClpXP proteases (in prokaryotes): Degradation machinery that targets misfolded or aggregated proteins.

Recognition and Management of Stalled Ribosomes:
Ltn1 (Listerin in eukaryotes): E3 ubiquitin ligase that tags the nascent polypeptide emerging from a stalled ribosome for degradation.
RQC complex: Recognizes stalled ribosomes and recruits Ltn1/Listerin.
Dom34 (Pelota in eukaryotes) and Hbs1: Recognize and rescue stalled ribosomes, facilitating ribosome recycling.

Post-Aggregation Repair and Refolding:
Hsp70 and Hsp40 (in eukaryotes): Chaperones that attempt to refold misfolded proteins.
DnaK and DnaJ (in prokaryotes): Analogous to Hsp70 and Hsp40, these chaperones aid in protein refolding.
Hsp100/Clp family: Disaggregases that solubilize aggregated proteins, making them accessible to refolding chaperones.

Targeting for Degradation:
Ubiquitination (in eukaryotes): Marks proteins for degradation by the proteasome.
ATP-dependent proteases (in prokaryotes): Recognize and degrade aberrant proteins without the need for prior tagging.

Cellular Stress Response:
Heat shock response: Upregulates chaperones and proteases in response to increased protein misfolding.
Unfolded protein response (in eukaryotes): Activated by the accumulation of misfolded proteins in the endoplasmic reticulum, leading to increased expression of chaperones and degradation machinery.

Through these intricate systems, cells ensure that proteins are correctly folded and functional, while misfolded or aggregated proteins are rapidly addressed, maintaining cellular health and homeostasis.

4. Discard and Recycling

Mechanisms that either discard faulty components or recycle them for future use.

Prokaryotic-Exclusive Mechanisms

tmRNA-Associated Ribosome Rescue

In bacteria, defective mRNAs that lack stop codons can cause ribosomes to stall during translation. To combat this, the tmRNA system acts as a rescue mechanism. It not only releases the stalled ribosome but also ensures that the partially synthesized polypeptide is tagged for degradation. This ensures the maintenance of protein synthesis fidelity and prevents the accumulation of potentially harmful incomplete proteins.

Reasons for Ribosome Stalling:
Defective mRNAs: mRNAs that lack proper stop codons can cause ribosomes to stall.
Secondary mRNA structures: These can impede ribosome progression.
Rare codons: Infrequently used codons might delay translation if the corresponding tRNA is scarce.
Damaged mRNA: Modifications or damages to mRNA can hinder ribosome movement.

Mechanism of tmRNA Action:
Recognition of Stalled Ribosomes: tmRNA, in association with SmpB protein, recognizes ribosomes that are stuck on defective mRNAs.
Trans-translation: tmRNA acts as both tRNA and mRNA. Its tRNA-like domain carries an alanine residue that is added to the growing polypeptide chain, while its mRNA-like domain provides a short open reading frame for continued translation.
Polypeptide Tagging: As translation proceeds on the tmRNA's open reading frame, a peptide tag is added to the C-terminus of the incomplete polypeptide, marking it for degradation.
Release of Stalled Ribosomes: After the tagging, the ribosome encounters a stop codon on the tmRNA, leading to the release of the ribosome and the tagged polypeptide.

Post-tmRNA Action:
Proteolysis: The tagged polypeptides are recognized by cellular proteases such as Lon and ClpXP, which degrade them.
Ribosome Recycling: Released ribosomes are recycled and made available for new rounds of translation.

Through this unique tmRNA-mediated rescue mechanism, bacteria ensure that stalled translation events are promptly resolved, maintaining the efficiency and fidelity of protein synthesis while preventing the build-up of truncated, non-functional proteins.

Ribosome Hibernation and Storage

Bacterial cells, when faced with unfavorable conditions, often employ adaptive mechanisms to conserve energy and ensure survival. One such strategy is ribosome hibernation, where ribosomes are rendered inactive to halt protein synthesis. This state of inactivity ensures cellular resources are preserved until conditions become favorable for growth and replication.

Reasons for Ribosome Hibernation:
Nutrient Limitation: Scarce resources can trigger cells to enter a dormant state.
Environmental Stress: Extreme temperatures, pH changes, or high salinity can stress cells.
Antibiotic Presence: Some antibiotics target the bacterial translation machinery, prompting hibernation as a protective response.
Starvation: Prolonged lack of essential nutrients can cause cells to hibernate their ribosomes.

Mechanism of Ribosome Hibernation:
Ribosome Dimerization: 70S ribosomes (comprising 50S and 30S subunits) associate to form inactive 100S dimers.
Hibernation Promoting Factor (HPF): This protein plays a pivotal role in ribosome dimerization in many bacteria.
Ribosome modulation factor (RMF): Found in some bacteria like E. coli, RMF aids in the formation of 90S dimers, which subsequently interact with another 70S ribosome to form the 100S dimer.
Inactivation of Anti-Association Factors: Certain factors that typically prevent ribosome subunit association are inactivated, further promoting dimerization.

Post-Hibernation Reactivation:
Environmental Sensing: Cells continuously sense environmental cues, primed to reactivate when conditions improve.
Ribosome Splitting: The 100S dimers dissociate back into individual 70S ribosomes, ready for translation.
Proteins like YhbH: In some bacteria, these proteins facilitate the dissociation of 100S dimers.
Resource Allocation: Upon reactivation, cellular resources are directed towards growth, replication, and protein synthesis.

Through the mechanism of ribosome hibernation, bacteria exhibit remarkable adaptability, allowing them to endure harsh conditions by temporarily shutting down a significant portion of their cellular machinery. Once the environment turns favorable, they quickly reactivate, resuming growth and replication.

Ribosome Rescue Pathways in Bacteria

Bacteria have specialized ribosome rescue systems to address situations where translation becomes stalled, often due to problematic mRNAs or environmental stressors. These pathways ensure that stalled ribosomes are efficiently recycled, preventing wastage of cellular resources and ensuring continued protein synthesis.

Reasons for Ribosome Stalling:
Truncated mRNAs: Absence of a stop codon can cause ribosomes to stall at the end of mRNA.
Secondary mRNA structures: These can impede ribosome movement along the mRNA.
Rare codons: Infrequently used codons can slow down translation if the corresponding tRNAs are in low supply.
Damaged or modified mRNA: Such mRNAs can interfere with normal translation dynamics.

Key Players in Ribosome Rescue:
ArfA (Alternative ribosome rescue factor A): In the absence of tmRNA, ArfA recognizes stalled ribosomes and recruits release factor RF2, promoting peptide release and ribosome recycling.
ArfB (Alternative ribosome rescue factor B): ArfB, also known as YaeJ, can directly recognize stalled ribosomes and has peptidyl-tRNA hydrolysis activity, facilitating the release of the nascent polypeptide chain.
tmRNA (transfer-messenger RNA): Acting with its partner protein SmpB, tmRNA performs a dual role. It first acts as a tRNA to add an alanine residue to the stalled peptide chain and then serves as an mRNA to provide a template for a short peptide tag. This tag targets the nascent protein for degradation by proteases.

Resolution of Stalled Ribosomes:
Ribosome Splitting: After the action of rescue factors, ribosomal subunits are separated for recycling.
mRNA Degradation: The problematic mRNA that caused the stall is targeted for degradation to prevent further stalling events.
Proteolysis: If the incomplete protein is tagged by tmRNA, proteases like Lon and ClpXP recognize and degrade the tagged protein.

These ribosome rescue pathways highlight the adaptability of bacteria, allowing them to quickly address and rectify translational stalls. By doing so, they ensure the optimal functioning of the translational machinery, a critical component of cellular survival and growth.

Ribosomal Stalling Quality Control in Prokaryotes

In prokaryotic cells, efficient protein synthesis is crucial for survival. When ribosomes stall during translation, the cell initiates specialized quality control pathways to address and resolve the issue. These mechanisms ensure the integrity of the translation process and maintain cellular homeostasis.

Reasons for Ribosome Stalling:
Defective mRNAs: Truncated or damaged mRNAs lacking stop codons can lead to ribosomal stalling.
Secondary structures: Complex structures in mRNAs can hinder the progression of ribosomes.
Amino acid starvation: A shortage of specific amino acids can slow down or halt translation.
Rare codon clusters: Sequences of infrequently used codons can impede translation due to limited tRNA availability.

Key Mechanisms and Players in Stalling Quality Control:
tmRNA system: In the presence of stalled ribosomes, transfer-messenger RNA (tmRNA) in association with SmpB acts to free the ribosome and tag the incomplete polypeptide for degradation.
ArfA and ArfB: These alternative ribosome rescue factors recognize stalled ribosomes and facilitate their release from problematic mRNAs. While ArfA works in conjunction with release factor RF2, ArfB possesses peptidyl-tRNA hydrolase activity.
RNase R: An exonuclease that degrades defective mRNAs causing ribosomal stalls.
Ribosome splitting: Factors like RRF (Ribosome Recycling Factor) and EF-G (Elongation Factor G) collaborate to dissociate stalled ribosomal complexes.

Actions Following Stalling Detection:
Ribosome recycling: After the stalled ribosome is identified, components are dissociated and recycled for new rounds of translation.
Degradation of faulty proteins: Incomplete polypeptides, especially those tagged by tmRNA, are targeted for degradation by proteases such as Lon and ClpXP.
mRNA turnover: mRNAs causing stalls are degraded to prevent repeated stalling events.

Through these intricate quality control pathways, prokaryotic cells ensure that translational stalls are quickly addressed, optimizing protein synthesis and minimizing the production of defective proteins. Collectively, these mechanisms are vital for bacterial survival and adaptation, especially in fluctuating environments. They ensure the optimal functioning of ribosomes, the key machinery of protein synthesis, and enable rapid responses to environmental challenges.

Eukaryotic-Exclusive Mechanisms

Ribosome Biogenesis Stress Response 

In eukaryotic cells, when there's an obstacle or hindrance in ribosome biogenesis, specific cellular responses get activated. These responses serve to ensure that ribosome production aligns with cellular needs and environmental conditions. Disturbances in ribosome biogenesis could result from various stressors like nutrient deficiency, DNA damage, or other cellular imbalances. The cell detects these disruptions and triggers a set of responses, often leading to a halt in cell cycle progression or activation of apoptosis in extreme cases. This mechanism ensures that only properly assembled ribosomes participate in protein synthesis, maintaining the fidelity of cellular functions.

Ribosome Biogenesis Stress Response in Eukaryotes

Eukaryotic cells possess intricate systems to monitor and maintain the integrity of ribosome biogenesis. Any disruptions in this process can lead to cellular imbalances and potential malfunctions. To address this, cells have a stress response mechanism, ensuring that ribosome production is fine-tuned according to cellular requirements and external conditions.

Causes of Ribosome Biogenesis Stress:
DNA Damage: Any damage to ribosomal DNA (rDNA) can hinder the transcription and processing of rRNA.
Nutrient Deficiency: Insufficient nutrients can impact the cellular energy levels, affecting ribosome assembly and function.
Protein Imbalances: Abnormal levels of ribosomal proteins or assembly factors can disrupt ribosome biogenesis.
Chemical Stressors: Certain chemicals or drugs can interfere with ribosomal components and their assembly.

Key Mechanisms and Players in Biogenesis Stress Response:
p53 pathway: One of the primary responders to ribosomal stress. Activation of p53 can lead to cell cycle arrest, allowing the cell time to repair or adapt to the stress.
Nucleolar surveillance: Detects and responds to disruptions in rRNA processing. Improperly processed rRNAs are rapidly degraded to prevent their incorporation into ribosomes.
Ribosomal protein-MDM2-p53 pathway: Certain ribosomal proteins (e.g., L5, L11) can bind to MDM2, an inhibitor of p53, leading to the stabilization and activation of p53.
c-Myc regulation: c-Myc, a transcription factor, plays a role in ribosome biogenesis. Under stress conditions, its levels and activity can be modulated to adjust ribosomal production.

Actions Following Biogenesis Stress Detection:
Cell Cycle Arrest: By halting the cell cycle, cells get an opportunity to repair any damages and restore homeostasis.
Apoptosis Activation: In extreme cases, where the stress is irreparable, the cell might activate programmed cell death to prevent the propagation of damaged cells.
Autophagy: Cellular degradation mechanism that can be activated to recycle damaged ribosomal components.
Adjustment of ribosomal gene transcription: Modulation of rDNA transcription rates to match the cellular conditions and needs.

By orchestrating these responses, eukaryotic cells ensure that ribosome biogenesis aligns with cellular demands, optimizing protein synthesis, and maintaining cellular health.

Discard and Recycling Extant in Both

Quality Control and Recycling of Ribosomes

Both prokaryotic and eukaryotic cells have dedicated systems to ensure the optimal functionality of their ribosomes. These mechanisms identify ribosomes that may be defective or have completed their translation cycle. Based on their condition, ribosomes are then either recycled back into the cellular machinery or targeted for degradation.

Reasons for Ribosome Quality Control:
Defective rRNA: Errors in rRNA processing or modifications can lead to non-functional ribosomes.
Stalled Translation: Ribosomes can become stalled on mRNAs, necessitating intervention.
Ageing of Ribosomes: Over time, ribosomal components can become worn out and less efficient.
Environmental Stressors: Factors like extreme temperatures or chemicals can impair ribosome function.

Mechanisms in Place for Ribosome Quality Control:
Ribosome rescue pathways: Specific proteins or factors, like ArfA, ArfB, and tmRNA in bacteria, intervene to free stalled ribosomes.
No-Go Decay (NGD) (eukaryotes): Targets mRNAs that lead to ribosome stalling for degradation.
Ribosome Splitting: Involves separating the two ribosomal subunits, allowing them to be recycled or degraded.

Processes Involved in Ribosome Recycling:
Rli1/ABCE1 (eukaryotes): An ATPase that plays a crucial role in splitting the ribosomal subunits after translation termination.
RRF and EF-G (prokaryotes): These factors work together to dissociate the ribosomal subunits, preparing them for the next round of translation.

Degradation Pathways:
Exoribonucleases: Enzymes that degrade RNA molecules, including defective rRNAs.
Proteasome (in eukaryotes): Complex that degrades damaged or unneeded proteins, including ribosomal proteins.
Lon and ClpXP proteases (in prokaryotes): These enzymes recognize and degrade damaged or unneeded ribosomal proteins.

By employing these mechanisms, both prokaryotic and eukaryotic cells maintain a pool of functional ribosomes, ensuring efficient protein synthesis and overall cellular health.

Ribosome Interactions with Autophagy Machinery

Autophagy is a cellular process designed to degrade and recycle cellular components, ensuring cellular health and homeostasis. Ribosomes, the cellular machinery responsible for protein synthesis, can interact with autophagy pathways, coordinating their functions and degradation processes.

Reasons for Ribosome-Autophagy Interactions:
Ribosomal Stress: Factors like nutrient deficiency, oxidative stress, or DNA damage can lead to impaired ribosomal function.
Defective Ribosomes: Ribosomes that are damaged or malfunctioning may need to be cleared from the cell.
Cellular Homeostasis: Maintaining a balance between protein synthesis and degradation is vital for cellular health.

Key Players in Ribosome-Autophagy Interactions:
LC3/Atg8: Proteins that are lipidated and play a central role in the formation of autophagosomes.
Atg1/ULK1 complex (in eukaryotes): Initiates the autophagy process in response to cellular signals.
RACK1: A ribosomal protein that can interact with autophagy machinery and is involved in signaling pathways.

Processes Involved in Ribosome Degradation by Autophagy:
Ribophagy: A specialized form of autophagy targeting ribosomes for degradation.
Endoplasmic Reticulum (ER) stress: Can induce autophagy, leading to the degradation of ribosomes associated with the ER.
Nucleophagy (in eukaryotes): The autophagic degradation of the nucleus, which includes rRNA and ribosomal components.

Benefits of Ribosome-Autophagy Interactions:
Cellular Recycling: Autophagy ensures that ribosomal components are recycled, providing building blocks for new ribosomes or other cellular components.
Quality Control: Damaged or old ribosomes are selectively targeted for degradation, ensuring optimal protein synthesis.
Response to Stress: Autophagy allows cells to adapt to changing environmental conditions by adjusting ribosome levels and protein synthesis rates.

In both prokaryotic and eukaryotic cells, the coordination between ribosomes and autophagy machinery ensures that the cell maintains a healthy balance of ribosomal components, allowing for efficient protein synthesis while also managing cellular stresses and damage.

No-Go Decay (NGD)

No-Go Decay is a specialized mRNA surveillance pathway in eukaryotic cells. It acts as a quality control mechanism, targeting mRNAs that cause ribosomal stalling, thereby ensuring efficient translation and protein synthesis.

Reasons for mRNA Targets by NGD:
Secondary Structures: Certain mRNAs may form complex structures that hinder ribosome movement.
Rare Codons: Sequences that are infrequently used can slow down or stall ribosomes due to the unavailability of corresponding tRNAs.
Damaged or Modified Bases: Modifications or damage to mRNA can interfere with the translation process.
Premature Stop Codons: These can result in truncated and non-functional proteins.

Key Players in No-Go Decay:
Upf1: An RNA helicase that identifies and binds to stalled ribosomes, initiating the NGD process.
Dom34 (in eukaryotes): A protein that recognizes stalled ribosomes and facilitates their dissociation.
Hbs1 (in eukaryotes): Works in conjunction with Dom34 to release stalled ribosomes.
Endonucleolytic cleavage: The mRNA is cleaved near the stalled ribosome, marking it for degradation.
Xrn1 (in eukaryotes): An exonuclease that degrades the cleaved mRNA fragments.

Processes Involved in No-Go Decay:
Recognition of Stalled Ribosomes: Factors like Dom34 and Hbs1 identify and bind to ribosomes that have been stalled during translation.
mRNA Cleavage: The mRNA is cleaved near the point of stalling.
mRNA Degradation: Cleaved mRNA fragments are degraded by exonucleases, preventing their further translation.

Benefits of No-Go Decay:
Quality Control: Ensures that only functional mRNAs are translated, preventing the production of non-functional or harmful proteins.
Protection: Prevents ribosomal traffic jams and potential damage to the cell by quickly addressing stalled translation.
Resource Allocation: By degrading problematic mRNAs, the cell can allocate resources to translating functional mRNAs.

The No-Go Decay pathway plays a critical role in maintaining the fidelity of protein synthesis, acting as a vital checkpoint to ensure cellular health and function.

Ribosome Rescue Pathways

Ribosome rescue pathways are vital mechanisms in both prokaryotic and eukaryotic cells, ensuring that ribosomes that become stuck on problematic mRNAs are efficiently addressed. These pathways either facilitate the resumption of translation or target the problematic mRNA and associated ribosome for degradation.

Reasons for Ribosomal Stalling:
Secondary Structures: mRNAs can form intricate structures that obstruct ribosome movement.
Rare Codons: Infrequently used sequences can slow down ribosomes if the corresponding tRNAs are not readily available.
Damaged or Modified Bases: Damage or modifications to mRNA bases can interfere with translation.
No Stop Codon: Ribosomes can stall if they reach the end of an mRNA without encountering a stop codon.

Key Players in Ribosome Rescue:
tmRNA (in prokaryotes): A specialized RNA molecule that acts to free the stalled ribosome and simultaneously tags the incomplete polypeptide for degradation.
ArfA and ArfB (in prokaryotes): Proteins that recognize stalled ribosomes and facilitate their release from problematic mRNAs.
Dom34 (in eukaryotes): A protein that identifies stalled ribosomes and helps dissociate them from the mRNA.
Hbs1 (in eukaryotes): Works with Dom34 to release stalled ribosomes.

Processes Involved in Ribosome Rescue:
Recognition of Stalled Ribosomes: Specific factors detect and bind to ribosomes that have been halted during translation.
Disassociation from mRNA: Stalled ribosomes are separated from the problematic mRNA, freeing them for new rounds of translation.
Degradation or Recycling: In some cases, the problematic mRNA is targeted for degradation. In other scenarios, the ribosome itself might be recycled or disassembled.

Benefits of Ribosome Rescue Pathways:
Maintaining Translation Efficiency: By swiftly addressing stalled ribosomes, cells ensure that the translation machinery remains productive.
Protecting Cellular Health: Preventing the accumulation of stalled ribosomes and potentially harmful incomplete proteins ensures cell vitality.
Resource Management: Efficient recycling of ribosomes and targeting problematic mRNAs for degradation conserves cellular resources.

Ribosome rescue pathways are essential for maintaining the smooth operation of the cellular translation machinery, ensuring that any interruptions in this critical process are promptly addressed.

Ribosome Collision and Quality Control

Ribosome collisions can occur when consecutive ribosomes translating the same mRNA run into each other, often due to problematic sequences or stalls in translation. Such collisions can be detrimental to cellular function, so both prokaryotic and eukaryotic cells have developed specific mechanisms to detect and resolve these events.

Reasons for Ribosome Collisions:
Stalling Sequences: Certain sequences in the mRNA can cause ribosomes to pause or stall, leading trailing ribosomes to catch up and collide.
Rare Codons: Sequences that code for infrequently used amino acids can slow down ribosomes if the corresponding tRNAs are scarce.
Secondary mRNA Structures: Complex structures in the mRNA can impede ribosome movement, causing backups.
Damaged mRNA: Breaks or modifications in the mRNA can halt ribosomes, leading to collisions.

Key Players in Ribosome Collision Response:
ZNF598 (in eukaryotes): A protein that recognizes collided ribosomes and ubiquitinates certain ribosomal proteins, signaling for the recruitment of other factors.
Hel2 (in eukaryotes): Works alongside ZNF598 to detect and mark collided ribosomes.
Rqc2 (in eukaryotes): A factor involved in ribosome-associated quality control, which aids in managing collided or stalled ribosomes.
tmRNA-ssrA System (in prokaryotes): Recognizes stalled ribosomes and tags the associated peptide for degradation.

Processes Involved in Ribosome Collision Response:
Detection of Collided Ribosomes: Specific cellular factors bind to collided ribosomes, distinguishing them from properly functioning ones.
Resolution or Recycling: Depending on the severity and cause of the collision, the ribosomes may be dissociated from the mRNA and recycled, or the problematic mRNA may be targeted for degradation.
Protein Quality Control: Incomplete proteins resulting from the collision might be tagged for degradation to prevent accumulation of non-functional polypeptides.

Benefits of Ribosome Collision Quality Control:
Maintaining Translation Efficiency: By addressing ribosome collisions promptly, cells ensure optimal utilization of the translation machinery.
Cellular Health: Preventing the buildup of collided ribosomes and potentially harmful proteins is crucial for cell vitality.
Resource Conservation: Efficiently managing ribosome collisions allows the cell to conserve energy and resources.

In conclusion, ribosome collision quality control is essential to ensure the smooth operation of the cellular translation machinery. Both prokaryotic and eukaryotic cells have dedicated systems to detect, manage, and resolve these potentially problematic events, underlining their importance in maintaining cellular homeostasis.

Stalled Ribosome Recognition

During translation, ribosomes can occasionally stall on mRNAs due to various reasons. Both prokaryotic and eukaryotic cells have set up systems to promptly detect these stalled ribosomes, ensuring that the translation process is either restarted or the ribosomes are targeted for degradation. Addressing stalled ribosomes is vital to maintain the efficiency of the translation machinery and to prevent the accumulation of incomplete polypeptides.

Reasons for Ribosome Stalling:
Problematic mRNA Sequences: Certain sequences can be challenging for ribosomes to read, causing them to pause.
Rare Codons: Infrequent codons may slow down translation if the appropriate tRNAs are not readily available.
Secondary mRNA Structures: Complex RNA structures can obstruct the ribosome's movement.
Damaged mRNA: Any modifications or disruptions in the mRNA can halt ribosome progression.

Key Players in Stalled Ribosome Detection:
ZNF598 (in eukaryotes): Recognizes and ubiquitinates specific ribosomal proteins on collided or stalled ribosomes.
Rqc2 (in eukaryotes): Involved in ribosome-associated quality control, it assists in handling stalled ribosomes.
tmRNA-ssrA System (in prokaryotes): Helps recognize and manage ribosomes that stall due to problematic mRNA sequences.
RelA and SpoT (in prokaryotes): These proteins sense ribosomal stalling and modulate the cellular response through the stringent response.

Processes Post Stalled Ribosome Recognition:
Ribosome Recycling: Factors dissociate the stalled ribosome from the mRNA, making it available for another round of translation.
mRNA Degradation: The problematic mRNA causing the stall might be degraded to prevent further issues.
Polypeptide Tagging: Incomplete proteins made due to ribosome stalling may be tagged for degradation.

Benefits of Stalled Ribosome Recognition:
Ensures Translation Efficiency: Detecting and addressing stalled ribosomes helps maintain a smooth translation process.
Cellular Health: Managing stalled ribosomes ensures that cells do not accumulate incomplete and potentially harmful proteins.
Resource Management: By recycling stalled ribosomes and degrading problematic mRNAs, cells optimize their resource utilization.

In summary, recognizing stalled ribosomes is a critical aspect of cellular quality control, ensuring the fidelity and efficiency of protein synthesis. Both prokaryotic and eukaryotic cells have sophisticated mechanisms to detect and manage these stalls, reflecting their significance in cellular function.

Translation Termination and Ribosome Recycling

Completing the translation of an mRNA molecule is a critical step in protein synthesis. To ensure that the synthesized protein is accurate and that the ribosomal machinery is available for subsequent rounds of translation, both prokaryotic and eukaryotic cells have established specific mechanisms for precise translation termination and efficient ribosome recycling.

Mechanisms of Translation Termination:
Release Factors (RFs): These proteins recognize stop codons on the mRNA and facilitate the release of the newly synthesized polypeptide from the ribosome.
Peptidyl Transferase Activity: The ribosome's peptidyl transferase center hydrolyzes the bond between the newly formed polypeptide and the tRNA, releasing the protein.

Ribosome Recycling Mechanisms:
Ribosome Recycling Factor (RRF) (in prokaryotes): This factor, in conjunction with elongation factor G (EF-G), helps dissociate the ribosomal subunits, making them available for a new round of translation.
eRF1 and eRF3 (in eukaryotes): These factors not only play a role in translation termination but also aid in the dissociation of ribosomal subunits.
ABCE1 (in eukaryotes): An ATPase that provides the energy needed to split the ribosomal subunits apart after translation termination.

Post-termination Processes:
mRNA Degradation: Once an mRNA molecule has been translated, it might be targeted for degradation to regulate protein synthesis.
tRNA Recharging: Used tRNAs are recharged with their specific amino acids by aminoacyl-tRNA synthetases, preparing them for future rounds of translation.

Benefits of Translation Termination and Ribosome Recycling:
Ensures Protein Accuracy: Proper termination ensures that proteins are synthesized according to their genetic blueprints.
Resource Efficiency: By recycling ribosomes, cells optimize their use of these intricate molecular machines.
Cellular Regulation: Proper termination and recycling processes allow cells to regulate protein synthesis in response to cellular needs and environmental conditions.

Translation termination and ribosome recycling are fundamental processes in cellular biology. Both prokaryotic and eukaryotic cells have mechanisms to ensure the fidelity of protein synthesis and the efficient reuse of ribosomes, underscoring the importance of these processes in maintaining cellular function and health.

mRNA Decay Pathways

To maintain cellular homeostasis and protect the integrity of protein synthesis, both prokaryotic and eukaryotic cells have mechanisms to regulate the abundance and quality of mRNAs. These mRNA decay pathways ensure that transcripts are present at appropriate levels and that faulty or no longer needed mRNAs are swiftly degraded.

Mechanisms of mRNA Decay:
5' to 3' Decay: This is a primary decay pathway where exonucleases degrade mRNAs starting from the 5' end.
3' to 5' Decay: In this pathway, the mRNA is degraded from its 3' end, often involving multi-protein complexes.

Specific Pathways and Factors:
Exosome Complex (in eukaryotes): A multi-protein complex that degrades mRNAs in the 3' to 5' direction.
Xrn1 (in eukaryotes): A 5' to 3' exonuclease responsible for degrading mRNAs after decapping.
Decapping Enzymes: These enzymes remove the 5' cap structure from mRNAs, making them susceptible to degradation.
RNase E and RNase R (in prokaryotes): Endoribonucleases that play roles in mRNA degradation.

Quality Control Mechanisms:
Nonsense-Mediated Decay (NMD) (in eukaryotes): Targets mRNAs containing premature stop codons for degradation, preventing the synthesis of truncated proteins.
No-Go Decay (NGD) (in eukaryotes): Targets mRNAs that cause ribosomal stalling for degradation.

Regulation and Significance:
Cellular Response to Stress: mRNA decay pathways can be modulated in response to cellular stress or environmental changes, ensuring that the appropriate gene expression profile is maintained.
Removal of Defective mRNAs: This ensures that only functional mRNAs are translated, thus maintaining the fidelity of protein synthesis.
Gene Expression Regulation: By controlling the lifespan of specific mRNAs, cells can finely tune their gene expression patterns in response to developmental cues or environmental signals.

In conclusion, mRNA decay pathways are crucial for cellular function and health. Both prokaryotic and eukaryotic cells have mechanisms to degrade mRNAs, ensuring that the gene expression landscape aligns with cellular needs and that potential threats from defective mRNAs are mitigated.

Translation Repression in Stress Response

To adapt to challenging environmental conditions and ensure survival, both prokaryotic and eukaryotic cells have developed mechanisms to regulate protein synthesis. One of these mechanisms involves the global suppression of translation, allowing the cell to conserve energy and prioritize vital processes.

Mechanisms of Translation Repression:
Phosphorylation of Translation Initiation Factors: Some cells modify translation initiation factors, making them inactive, thus inhibiting the initiation of translation.
Sequestration of Translation Machinery: In certain stress conditions, ribosomes and other translation components can be sequestered into specific cellular compartments or structures, preventing them from participating in translation.

Specific Pathways and Factors:
eIF2α Phosphorylation (in eukaryotes): In response to various stresses, eIF2α can be phosphorylated, leading to a decrease in general protein synthesis while allowing the translation of specific mRNAs.
RelA/SpoT System (in prokaryotes): In response to amino acid starvation, these enzymes synthesize (p)ppGpp, a molecule that represses translation by binding to the ribosome.
RNA-Binding Proteins: Certain proteins can bind to mRNAs and prevent their translation in stress conditions.

Types of Stressors:
Nutrient Starvation: Lack of essential nutrients can trigger translation repression as the cell conserves resources.
Oxidative Stress: High levels of reactive oxygen species can damage cellular components, leading to a reduction in translation.
Heat Shock: Elevated temperatures can denature proteins, prompting cells to halt new protein synthesis and focus on protein refolding or degradation.

Cellular Outcomes:
Conservation of Energy: By repressing translation, cells can save energy, which can be redirected to essential survival processes.
Protein Homeostasis: Reducing translation helps maintain the balance of proteins, preventing aggregation or misfolding.
Preparation for Recovery: By halting translation, cells can prepare to quickly resume growth and replication once the stressor is removed.

Translation repression during stress response is a critical adaptive mechanism. Both prokaryotic and eukaryotic cells utilize various strategies to inhibit protein synthesis, ensuring cell survival and proper function under adverse conditions.

5. Post-translation Quality Control

Post-translational modifications and quality checks are critical for ensuring that proteins are functional, properly folded, and localized, and that any misfolded or improperly synthesized proteins are recognized and degraded.

Eukaryotic-Exclusive Mechanisms

Regulation of Ribosome Biogenesis in Development

As eukaryotic cells progress through different stages of development, the rate and regulation of ribosome production can vary to match the cellular requirements of each phase.
Differentiation: As cells differentiate into specific cell types, they adjust ribosome production to cater to specialized protein synthesis needs.
Growth Factors: The presence or absence of certain growth factors can influence ribosome biogenesis, impacting cell growth and proliferation.
Cell Cycle: During specific stages of the cell cycle, particularly in preparation for cell division, there's an upsurge in ribosome production to meet increased protein synthesis demands.

Coordination with Other Cellular Processes

For optimal function and efficiency, eukaryotic cells synchronize ribosome biogenesis with various cellular events and pathways. This coordination ensures that ribosome production aligns with the cell's metabolic state, growth conditions, and other cellular demands, enabling a balanced and harmonized cellular response.

Mechanisms of Coordination:
Cell Cycle Regulation: Ribosome biogenesis is often synchronized with the cell cycle, ensuring that rapidly dividing cells have adequate ribosomes for protein synthesis.
Nutrient Sensing: Cells adjust ribosome production based on nutrient availability. For example, during nutrient abundance, ribosome biogenesis may be upregulated to support increased protein synthesis.
Stress Response: In conditions of cellular stress, ribosome biogenesis might be modulated to prioritize repair and maintenance pathways.

Interplay with Other Processes:
DNA Replication: Ribosome biogenesis is intricately linked to DNA replication, ensuring that there are enough ribosomes to meet the protein synthesis needs of daughter cells post-division.
mRNA Translation: The rate of ribosome production can influence mRNA translation efficiency. Cells might upregulate ribosome biogenesis when there's a need for enhanced protein synthesis.
Energy Metabolism: Ribosome production and function are energy-intensive. Coordination with metabolic pathways ensures that energy supply aligns with the demands of ribosome biogenesis.

Feedback Mechanisms:
Quality Control: Errors in ribosome assembly or function can trigger feedback mechanisms that reduce ribosome biogenesis until the errors are rectified.
Signaling Pathways: Cellular signaling pathways, like the mTOR pathway, can influence ribosome biogenesis based on external and internal cues, ensuring adaptive responses to changing conditions.

Nutrient Sensing and Ribosome Production

The ability of cells to sense and respond to nutrient availability is crucial for maintaining cellular health and ensuring efficient use of resources. By modulating ribosome production based on nutrient levels, cells can optimize protein synthesis and conserve energy and materials.

Mechanisms of Nutrient Sensing:
mTOR Pathway (in eukaryotes): One of the central regulators of cell growth and metabolism, the mTOR pathway integrates signals from nutrients, especially amino acids, to regulate ribosome biogenesis and protein synthesis.
AMPK Activation: During low energy conditions, AMPK (AMP-activated protein kinase) is activated, leading to the inhibition of processes that consume energy, including ribosome production.
Guanosine Tetra- and Pentaphosphate (ppGpp) in Prokaryotes: In response to amino acid starvation, bacterial cells synthesize ppGpp, which acts as an alarmone, reducing ribosome biogenesis and redirecting resources to survival pathways.

Adaptive Responses to Nutrient Scarcity:
Ribosome Downregulation: During nutrient scarcity, cells reduce the overall rate of ribosome production, thereby conserving resources and reducing energy expenditure on protein synthesis.
Selective mRNA Translation: Under nutrient-limited conditions, cells can prioritize the translation of specific mRNAs that encode proteins essential for survival and stress response.
Autophagy: When nutrients are scarce, cells may upregulate autophagy, a process where they degrade and recycle cellular components, including ribosomes, to obtain essential building blocks and energy.

Recovery and Upregulation:
Once nutrient levels are restored, cells rapidly upregulate ribosome biogenesis to meet the increased demand for protein synthesis, ensuring growth and proliferation in a favorable environment.

Stress Response: In situations of cellular stress, ribosome biogenesis might be downregulated to conserve energy and prioritize repair mechanisms.

Stress Response and Ribosome Biogenesis

The ability of cells to swiftly react to various stressors ensures their survival and optimal function. By adjusting the rate of ribosome production, cells can conserve energy, redirect resources, and prioritize cellular repair mechanisms over protein synthesis.

Mechanisms of Stress-Induced Regulation:
mTOR Inhibition (in eukaryotes): Under stress conditions, the mTOR signaling pathway, which promotes ribosome biogenesis under normal conditions, might be inhibited, leading to reduced ribosome production.
Activation of eIF2α: Phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) inhibits general protein synthesis but promotes translation of specific mRNAs that help cells cope with stress.
Heat Shock Response: In response to elevated temperatures, cells might produce heat shock proteins that stabilize existing proteins and can lead to a temporary reduction in ribosome biogenesis.

Adaptive Responses to Stress:
Conservation of Energy: By downregulating ribosome production, cells can conserve energy, which can then be redirected to essential survival pathways.
Prioritization of Repair Mechanisms: Cells might prioritize DNA repair, detoxification of reactive oxygen species, and other protective mechanisms over protein synthesis.
Preparation for Recovery: By modulating ribosome biogenesis during stress, cells can quickly resume growth and protein synthesis once the stressor is alleviated.

Ribosome Biogenesis in Aging

As organisms progress through their lifespan, cellular processes, including ribosome biogenesis, undergo changes. Eukaryotic cells have developed mechanisms to adjust ribosome synthesis in response to the challenges and requirements of aging, ensuring optimal cellular function and adaptation to the aging process.

Mechanisms Influencing Ribosome Production in Aging:
Decline in Ribosomal RNA Synthesis: As cells age, there might be a reduction in the synthesis of ribosomal RNA, leading to decreased ribosome production.
Changes in Ribosomal Proteins: The expression levels of certain ribosomal proteins might change with age, affecting the assembly and function of ribosomes.
DNA Damage Response: Accumulation of DNA damage over time can influence the transcription of ribosomal genes, impacting ribosome biogenesis.

Effects of Altered Ribosome Biogenesis in Aging:
Reduced Protein Synthesis: A decline in ribosome production can lead to a reduced capacity for protein synthesis, affecting cellular function and repair mechanisms.
Cellular Senescence: Changes in ribosome biogenesis can contribute to the entry of cells into a state of senescence, where they remain metabolically active but cease to divide.
Longevity Pathways: Some studies suggest that modulating ribosome biogenesis might activate pathways associated with longevity, potentially extending lifespan.

Adaptive Responses in Aging:
Selective mRNA Translation: Aged cells might prioritize the translation of specific mRNAs that support adaptation to the challenges of aging.
Upregulation of Repair Pathways: Cells might enhance DNA repair and protein quality control mechanisms to cope with the increased challenges faced during aging.

Ribosome Synthesis and Cellular Senescence

Cellular senescence is a state where cells lose their ability to divide and proliferate, often in response to various stressors or as a natural part of aging. Eukaryotic cells adjust ribosome synthesis in senescent cells, ensuring that protein production aligns with the altered metabolic and functional state of these cells.

Mechanisms Influencing Ribosome Synthesis in Senescence:
Reduced rRNA Transcription: Senescent cells often exhibit reduced transcription of ribosomal RNA, leading to decreased ribosome synthesis.
Altered Ribosomal Protein Expression: The expression of certain ribosomal proteins may be downregulated in senescent cells, affecting ribosome assembly and function.
DNA Damage Response: DNA damage, a trigger for cellular senescence, can influence ribosome biogenesis by affecting rRNA transcription and ribosomal protein synthesis.

Implications of Altered Ribosome Synthesis:
Decreased Protein Synthesis: With fewer ribosomes, senescent cells have a reduced capacity for protein synthesis, contributing to their altered functional state.
Quality Control: Senescent cells may have enhanced quality control mechanisms, ensuring that the limited ribosomes produced are functional and error-free.
Energy Conservation: By reducing ribosome synthesis, senescent cells can conserve energy, redirecting it to maintenance and repair pathways.

Adaptive Responses in Senescence:
Stress Response Pathways: Senescent cells activate various stress response pathways, which might further modulate ribosome biogenesis to prioritize survival and cellular integrity.
Autophagy and Proteostasis: With limited protein synthesis, senescent cells rely more on autophagy and other proteostasis mechanisms to maintain cellular protein balance and functionality.

Ribosome Biogenesis and Apoptosis

Apoptosis, or programmed cell death, is a regulated process that plays a pivotal role in maintaining tissue homeostasis and eliminating damaged or harmful cells. The intricate relationship between ribosome biogenesis and apoptosis underscores the balance that eukaryotic cells must strike between growth, survival, and death.

Mechanisms Linking Ribosome Biogenesis and Apoptosis:
p53 Activation: DNA damage and disruptions in ribosome biogenesis can activate the p53 pathway. Elevated p53 can halt ribosome biogenesis and trigger apoptotic pathways.
Nucleolar Stress: Disturbances in ribosome production can lead to nucleolar stress, which can either halt cell growth or initiate apoptosis, depending on the severity and duration of the stress.
rRNA Processing: Defects in rRNA processing and assembly can act as signals for apoptosis initiation, ensuring that cells with compromised ribosomes do not proliferate.

Implications of Altered Ribosome Synthesis:
Cell Cycle Arrest: Before committing to apoptosis, cells may first undergo cell cycle arrest, providing an opportunity for repair mechanisms to act.
Quality Control: Cells have enhanced quality control mechanisms to ensure ribosomal components are correctly processed. Failure in these checks can tip the balance towards apoptosis.
Energy Dynamics: A decrease in ribosome biogenesis could lead to energy conservation, potentially delaying apoptosis onset. However, persistent energy deficits might accelerate the apoptotic process.

Adaptive Responses to Apoptotic Signals:
Survival Pathways: Cells can activate survival pathways in response to mild disruptions in ribosome biogenesis, attempting to restore normalcy.
Autophagy: Autophagy, a cellular recycling process, can be upregulated in response to disruptions in ribosome production, potentially providing resources or acting as a temporary survival mechanism.

Ribosome Interactions with Cellular Organelles

Ribosome Interactions with Endosomes and Lysosomes

Endosomes and lysosomes are pivotal components of the endocytic pathway, handling the internalization and degradation of extracellular materials and damaged cellular components. The coordination between ribosomes and these organelles ensures seamless cellular homeostasis and optimal protein synthesis.

Mechanisms of Interaction:
Ribosome Localization: Ribosomes can be localized near endosomes and lysosomes, facilitating the translation of membrane proteins and proteins destined for these organelles.
Signal Recognition Particle (SRP): SRP can guide the ribosome-nascent chain complexes to the endosomal or lysosomal membrane, ensuring efficient protein targeting.
Lysosome Biogenesis: Ribosomes play a crucial role in the synthesis of lysosomal enzymes and membrane proteins, aiding in the formation and function of lysosomes.

Functional Implications:
Protein Trafficking: The proximity of ribosomes to endosomes and lysosomes can enhance the trafficking of newly synthesized proteins to these organelles, ensuring their functionality.
Cellular Response to Stress: Under certain stress conditions, ribosomes can alter their interactions with endosomes and lysosomes, influencing cellular degradation pathways and homeostasis.
Autophagy: Ribosomes coordinate with lysosomes during autophagy, a process where cells degrade and recycle their components. Proper ribosomal function ensures efficient autophagosome formation and lysosomal degradation.

Adaptive Mechanisms:
Quality Control: Cells have mechanisms to ensure that proteins synthesized by ribosomes and destined for endosomes or lysosomes are properly folded and functional. Misfolded proteins can be directed for degradation.
Endosomal Recycling: Ribosomes can influence the recycling of endosomal components, ensuring that essential membrane proteins are returned to the cell surface or other organelles.

Ribosome Interactions with the Nuclear Envelope

The nuclear envelope, a double membrane structure that encloses the nucleus, plays a critical role in separating the nuclear contents from the cytoplasm in eukaryotic cells. Ribosomes, the protein synthesis machinery of the cell, can have intriguing interactions with the nuclear envelope, which can influence various cellular processes.

Mechanisms of Interaction:
Ribosome Biogenesis: The initial stages of ribosome assembly occur in the nucleolus, a region within the nucleus. Once preliminary assembly is complete, pre-ribosomal particles exit the nucleus through nuclear pore complexes, which span the nuclear envelope.
Synthesis of Membrane Proteins: Ribosomes translating mRNAs that encode integral membrane proteins can be tethered to the cytoplasmic face of the nuclear envelope. This ensures efficient integration of these proteins into the endoplasmic reticulum (ER), which is continuous with the nuclear envelope.
Signal Recognition Particle (SRP): The SRP can bind to ribosomes translating specific mRNAs, guiding them to the ER membrane, which is in close proximity to the nuclear envelope.

Functional Implications:
Coordination with the ER: Given the continuity of the nuclear envelope with the ER, ribosomes on the nuclear envelope can contribute to the synthesis of proteins that get folded and modified in the ER lumen.
Nuclear-Cytoplasmic Transport: Ribosomes near the nuclear envelope may facilitate efficient transport of specific mRNAs or proteins between the nucleus and cytoplasm.
Cell Cycle Regulation: Interactions between ribosomes and the nuclear envelope can influence cell cycle progression, especially during phases where the nuclear envelope undergoes reorganization.

Adaptive Mechanisms:
Response to Stress: Under certain stress conditions, ribosomes might modify their interactions with the nuclear envelope, potentially influencing gene expression patterns and protein synthesis rates.
Quality Control: Ribosomes near the nuclear envelope may play a role in the quality control of newly synthesized proteins, ensuring that only properly folded and functional proteins proceed through the secretory pathway.

Ribosome Interactions with the Golgi Apparatus

The Golgi apparatus, often referred to as the cell's "post office," is responsible for the modification, sorting, and packaging of proteins and lipids for transport to various destinations within or outside the cell. Ribosomes, which are responsible for protein synthesis, can have specific interactions with the Golgi apparatus, ensuring efficient processing and transport of proteins.

Mechanisms of Interaction:
Endoplasmic Reticulum (ER) to Golgi Transport: After ribosomes synthesize proteins in the ER, these proteins are packaged into vesicles that bud off from the ER and are transported to the Golgi apparatus for further modification.
Synthesis of Golgi-Resident Proteins: Certain proteins that function within the Golgi apparatus itself are synthesized by ribosomes and then specifically targeted and transported to the Golgi.
Membrane Protein Synthesis: Ribosomes translating mRNAs that encode integral membrane proteins destined for the Golgi can be tethered to the ER, ensuring their correct localization after synthesis.

Functional Implications:
Post-translational Modifications: Proteins synthesized by ribosomes and destined for the Golgi undergo various post-translational modifications, such as glycosylation, which are critical for their function and localization.
Protein Sorting and Secretion: After modification in the Golgi, proteins are sorted and packaged into vesicles for transport to their final destinations, be it other cellular compartments, the cell surface, or secretion out of the cell.
Quality Control: Interactions between ribosomes and the Golgi apparatus can play a role in the quality control of newly synthesized proteins. Misfolded or improperly modified proteins can be recognized and targeted for degradation.

Adaptive Mechanisms:
Response to Cellular Needs: The nature and extent of ribosome interactions with the Golgi apparatus can be modulated based on the cell's needs. For instance, during high secretory activity, there might be increased coordination between ribosomes and the Golgi.
Stress Response: Cellular stress can lead to alterations in protein synthesis and processing. Ribosomes may modulate their interactions with the Golgi under such conditions, influencing the overall protein output and quality.

Environmental Responses and Ribosome Synthesis

Ribosome Synthesis in Response to Hypoxia 

Hypoxia, or reduced oxygen availability, is a condition that can arise due to various reasons, including cellular growth in a dense tissue matrix, vascular abnormalities, or high altitudes. Eukaryotic cells have developed mechanisms to adjust ribosome synthesis in response to hypoxia, ensuring that protein production is modulated in line with the available oxygen levels and metabolic needs.

Mechanisms of Adaptation:
Hypoxia-Inducible Factors (HIFs): One of the primary responders to low oxygen levels, HIFs are transcription factors that get stabilized under hypoxic conditions. They regulate the expression of genes involved in multiple cellular processes, including ribosome biogenesis.
mTOR Pathway Modulation: The mTOR pathway, which is a central regulator of cell growth and metabolism, can be inhibited under hypoxic conditions. This inhibition leads to a reduction in ribosome synthesis and protein translation.
rRNA Transcriptional Changes: Hypoxia can lead to alterations in the transcription of ribosomal RNA (rRNA) genes, which form the core of ribosomes. Reduced rRNA synthesis can lead to fewer ribosomes being produced.

Functional Implications:
Energy Conservation: By reducing ribosome synthesis and subsequent protein translation, cells conserve energy, which is crucial under conditions where oxygen-dependent ATP production might be compromised.
Selective mRNA Translation: While general protein synthesis might be reduced, cells under hypoxia can prioritize the translation of specific mRNAs that encode proteins vital for survival and adaptation to low oxygen levels.
Cell Survival and Proliferation: Adjustments in ribosome synthesis can influence cell cycle progression and proliferation rates. In some contexts, hypoxia might lead to a temporary halt in cell division until conditions normalize.

Recovery Mechanisms:
Oxygen Sensing Mechanisms: When oxygen levels are restored, cells can rapidly sense this change and upregulate ribosome biogenesis to meet the increased demand for protein synthesis. This ensures a swift return to normal cellular function and growth.
Degradation of HIFs: Once oxygen levels normalize, HIFs are targeted for degradation, thus relieving their inhibitory effects on ribosome synthesis and other processes.

Ribosome Biogenesis in Response to Radiation

Radiation, whether ionizing or ultraviolet, poses significant threats to cellular integrity, DNA stability, and overall cell function. Eukaryotic cells, when exposed to such radiation, often undergo various adaptive and protective changes, including alterations in ribosome synthesis. These changes ensure that the cell can either repair the damage, pause cellular processes until recovery, or initiate programmed cell death if the damage is beyond repair.

Mechanisms of Response:
DNA Damage Response (DDR): Radiation primarily damages DNA. The DDR gets activated upon detecting DNA lesions, leading to cell cycle arrest, DNA repair, or apoptosis. Since ribosome biogenesis is closely linked to the cell cycle, the DDR can indirectly influence ribosomal RNA (rRNA) synthesis.
p53 Activation: p53 is a key tumor suppressor protein that gets stabilized and activated upon DNA damage. Activated p53 can repress rRNA transcription by interacting with the transcription machinery of the ribosomal genes.
mTOR Pathway Modulation: Radiation can lead to oxidative stress, and this, in turn, can inhibit the mTOR pathway, which is central to cell growth and ribosome synthesis.

Functional Implications:
Energy Conservation: By modulating ribosome synthesis, cells can conserve energy, redirecting it towards repair mechanisms.
Cell Cycle Arrest: Reduction in ribosome synthesis can halt the cell cycle, allowing time for DNA repair.
Selective mRNA Translation: Cells might prioritize the translation of specific mRNAs encoding proteins that are crucial for DNA repair, stress response, and survival under radiative stress.

Recovery Mechanisms:
Checkpoints Release: Once DNA repair is successful, cell cycle checkpoints are released, and ribosome biogenesis is upregulated.
Antioxidant Defense Upregulation: To combat oxidative stress induced by radiation, cells might enhance their antioxidant defenses. As the oxidative stress subsides, the mTOR pathway can be reactivated, promoting ribosome synthesis.

Ribosome Regulation and Interaction with Cellular Processes

mRNA Surveillance and Exon Junction Complexes (EJCs)

The accurate splicing of pre-mRNA is vital for ensuring the correct expression of genes. Mistakes in this process can lead to non-functional proteins or proteins with altered functions, potentially leading to cellular dysfunction or diseases. Eukaryotic cells employ various surveillance mechanisms to identify and address splicing errors, and the Exon Junction Complex (EJC) plays a pivotal role in this context.

Roles of EJCs:
Splice Site Marking: EJCs are deposited 20-24 nucleotides upstream of exon-exon junctions. This marking acts as a "proof of splicing" and can be recognized by other cellular machinery.
Nonsense-Mediated Decay (NMD): EJCs collaborate with the NMD pathway to degrade mRNAs containing premature stop codons. If a ribosome encounters a stop codon while an EJC is still bound to the mRNA, it's an indication that the stop codon is premature, triggering mRNA degradation.
mRNA Transport: EJCs facilitate the export of mature mRNAs from the nucleus to the cytoplasm, ensuring that only correctly spliced mRNAs reach the translation machinery.
Translation Enhancement: EJCs can enhance the efficiency of translation initiation, ensuring that mRNAs are effectively used for protein synthesis.

Functional Implications:
Genome Integrity: By marking spliced mRNAs, EJCs contribute to maintaining genome integrity by ensuring only correctly processed mRNAs are translated.
Cellular Homeostasis: Efficient mRNA surveillance and degradation of faulty mRNAs prevent the synthesis of non-functional or harmful proteins.
Response to Cellular Stress: During cellular stress, the EJC-NMD pathway can be modulated to adjust protein synthesis according to cellular needs.

Evolutionary Significance:
Complexity of Eukaryotic Genomes: The presence of introns in eukaryotic genes necessitates intricate splicing mechanisms. EJCs have evolved to manage the complexity of eukaryotic mRNA processing.

Regulation by Small Molecules and Drugs 

The ribosome, being the cellular machinery responsible for protein synthesis, is a primary target for many therapeutic interventions in eukaryotic cells. Small molecules and drugs can interact with ribosomes to either enhance or inhibit their function, depending on the desired therapeutic outcome.

Mechanisms of Action:
Ribosome Targeting Antibiotics: Many antibiotics, such as tetracyclines and macrolides, bind specifically to the ribosomes of bacteria, inhibiting protein synthesis and thus bacterial growth. Although these primarily target prokaryotic ribosomes, some can have off-target effects on eukaryotic cells.
Antitumor Agents: Certain anticancer drugs, like homoharringtonine, interfere with the ribosome's function in eukaryotic cells, leading to decreased protein synthesis and growth inhibition of cancer cells.
Neurological Drugs: Some molecules affecting ribosome function have been explored for treating neurological disorders by modulating protein synthesis in neurons.
Antiviral Agents: Ribosome-targeting antiviral drugs can inhibit the synthesis of viral proteins, preventing the replication and spread of viruses within the host.

Effects on Cellular Function:
Cell Growth and Proliferation: By regulating ribosome function, these molecules can control cell growth, making them valuable tools in cancer therapy.
Cellular Stress Response: Cells can sense and respond to the inhibition of protein synthesis by activating stress pathways, potentially leading to cell cycle arrest or apoptosis.
Immune Response Modulation: Some drugs affecting ribosome function can modulate the immune response, either enhancing or suppressing it based on the therapeutic need.

Research and Therapeutic Implications:
Drug Development: Understanding the interaction of drugs with ribosomes opens avenues for developing novel therapeutic agents targeting ribosome function.
Side Effects: As ribosomes are essential for cellular function, drugs targeting them can have side effects, making it crucial to design specific and targeted interventions.
Personalized Medicine: Given the variability in ribosomal proteins and rRNA sequences among individuals, personalized approaches can be employed to optimize therapeutic outcomes when targeting ribosomes.

Coordination with Cell Cycle Progression 

The synthesis and function of ribosomes are intimately linked with the cell cycle, ensuring that protein synthesis is attuned to the cellular requirements at various stages of growth and division.

Synchronization with Cell Cycle Phases:
G1 Phase: As cells prepare for DNA replication, there's an upregulation in ribosome biogenesis to meet the increased protein synthesis demand in the upcoming S phase.
S Phase: During DNA replication, ribosome synthesis continues at a steady rate, ensuring a constant supply of proteins required for DNA synthesis and repair mechanisms.
G2 Phase: Prior to mitosis, cells might modulate ribosome synthesis to conserve energy and resources for the upcoming cell division.
M Phase (Mitosis): During cell division, ribosome synthesis is generally reduced as the cell's machinery is primarily focused on the segregation of chromosomes and organelles.

Cell Cycle Checkpoints:
G1/S Checkpoint: The cell ensures that all necessary proteins, synthesized by ribosomes, are present before committing to DNA replication.
G2/M Checkpoint: Proper synthesis and function of ribosomes are checked before the cell enters mitosis.
Spindle Assembly Checkpoint: Although primarily concerned with chromosome alignment, the correct functioning of ribosomes is essential for synthesizing proteins that oversee this checkpoint.

Regulation Mechanisms:
Cyclin-Dependent Kinases (CDKs): These enzymes, which regulate the cell cycle, might also influence ribosome synthesis and function.
mTOR Pathway: A central regulator of cell growth and metabolism, the mTOR pathway can integrate signals from growth factors and nutrients to regulate ribosome biogenesis and protein synthesis.
p53 Protein: This tumor suppressor protein can halt cell cycle progression under stress conditions and might influence ribosome synthesis.

Implications:
Cell Growth and Proliferation: By aligning ribosome synthesis with cell cycle progression, cells ensure optimal growth and division.
Cellular Stress Response: Any disruption in ribosome function can activate stress responses, potentially leading to cell cycle arrest or apoptosis.
Therapeutic Interventions: Targeting ribosome synthesis and function can influence cell cycle progression, making it a potential strategy in cancer therapy.

Ribosome Biogenesis in Stem Cells

Stem cells have the unique ability to differentiate into various cell types and maintain tissue homeostasis. The synthesis and function of ribosomes in stem cells are essential not only for their proliferative capacity but also for their potential to differentiate.

Role of Ribosomes in Stem Cells:
Stem cells have a high demand for protein synthesis due to their rapid proliferation and potential to differentiate. Proper ribosome function ensures that these cells can meet their protein synthesis requirements.
Stem Cell Renewal:
For stem cells to maintain their undifferentiated state, specific sets of proteins need to be synthesized. Ribosomes play a vital role in producing these proteins, ensuring the stem cells' self-renewal capacity.
Differentiation:
As stem cells differentiate, their protein synthesis demands change. Ribosome biogenesis adjusts to these changing needs, ensuring that the right proteins are synthesized to aid in the differentiation process.
Ribosomal Proteins and Stem Cell Fate:
Some ribosomal proteins have been implicated in influencing stem cell fate decisions, either promoting self-renewal or triggering differentiation.
Stress Response in Stem Cells:
Given their long lifespan, stem cells need to efficiently manage cellular stress. Proper ribosome function is crucial in this regard, as any disruption in protein synthesis can affect stem cell function and viability.
Therapeutic Potential:
Understanding ribosome biogenesis in stem cells can offer insights into stem cell function and provide avenues for therapeutic interventions, especially in regenerative medicine.

Implications:
Tissue Regeneration: Stem cells play a crucial role in tissue repair and regeneration. Ensuring proper ribosome function in these cells can enhance regenerative potential.
Disease Modeling: Stem cells are increasingly used for disease modeling. Insights into ribosome biogenesis can help in understanding disease mechanisms at the cellular level.
Cancer Stem Cells: Cancer stem cells are a subset of tumor cells with stem-like properties. Understanding ribosome biogenesis in these cells can provide therapeutic targets for cancer treatment.

Ribosome Biogenesis and Stress Responses

Ribosome Biogenesis and DNA Damage Response

Stressful conditions can challenge the integrity and functionality of cellular machinery. Eukaryotic cells have mechanisms that synchronize ribosome biogenesis with their stress responses to ensure that protein synthesis remains accurate and efficient during these times.

Role in Error Check and Repair:
When cells encounter stress, there's an increased likelihood of errors in protein synthesis. Ribosome biogenesis plays a role in monitoring and correcting these errors. A tight coordination ensures that malfunctioning ribosomes are not produced, which could further exacerbate the cellular stress.
Ribosome Biogenesis and DNA Damage Response:
DNA damage can lead to errors in transcription, which can subsequently affect ribosome biogenesis and protein synthesis. Eukaryotic cells have mechanisms that synchronize ribosome synthesis with the DNA damage response to prevent the production of faulty ribosomes.
p53 Activation: One of the primary responders to DNA damage, p53, not only halts the cell cycle but also modulates ribosome biogenesis. This ensures that damaged DNA doesn't lead to the production of malfunctioning ribosomes.
Nucleolar Stress: Nucleoli, the site of ribosome biogenesis, can sense DNA damage and trigger a stress response. This halts ribosome production, allowing the cell time to repair the DNA damage and prevent the synthesis of faulty ribosomes.
Recycling and Degradation:
If the ribosome biogenesis machinery produces faulty ribosomes, cells have pathways to either repair or degrade them. This ensures that only functional ribosomes are active in protein synthesis.
Nucleophagy: A process where cells degrade excess or damaged nucleoli, ensuring that faulty ribosome production machinery is removed.
Ribophagy: The selective degradation of ribosomes. In times of stress or when ribosomes are damaged, this process is upregulated to maintain cellular homeostasis.
Implications for Cellular Health:
Ensuring proper ribosome biogenesis during stress conditions and DNA damage responses is crucial for cellular health. Faulty ribosomes can lead to widespread protein misfolding and dysfunction. By coordinating ribosome synthesis with stress responses, cells maintain their protein homeostasis and overall health.

Ribosome Biogenesis and DNA Replication Stress

DNA replication stress, which arises from obstacles during the DNA copying process, can impact various cellular functions, including ribosome biogenesis. Eukaryotic cells have established mechanisms to ensure that ribosome production is aligned with the challenges posed by replication stress, thereby maintaining cellular equilibrium and functionality.

Role in Error Check and Repair:
During DNA replication stress, there's an increased risk of errors in the transcription of rRNA genes and subsequent ribosome assembly. To counteract this, ribosome biogenesis is equipped to detect and rectify these inconsistencies. By doing so, the cell prevents the creation of non-functional ribosomes that could exacerbate the replication stress.
Ribosome Biogenesis and Replication Stress Response:
DNA replication stress can affect the integrity of rRNA genes and their transcription rates. As a result, cells have mechanisms to modulate ribosome synthesis in response to these challenges, ensuring that ribosome production does not outpace the cell's current capacity.
ATR Activation: A pivotal responder to replication stress, ATR (Ataxia Telangiectasia and Rad3-related protein) not only manages the replication stress response but also impacts ribosome biogenesis. By doing so, it ensures that replication stress does not lead to the unchecked production of potentially defective ribosomes.
Nucleolar Response: The nucleolus, where ribosome biogenesis primarily takes place, can sense replication stress and adjust its activities accordingly. This might involve slowing down rRNA transcription or modifying ribosomal protein synthesis to align with the current cellular state.
Recycling and Degradation:
Should replication stress lead to the production of flawed ribosomes, cells have mechanisms in place to either refurbish or dismantle them. This ensures the availability of functional ribosomes and prevents the buildup of defective ones.
RER (Ribosome Export Response): In cases of replication stress, ribosome export from the nucleolus to the cytoplasm can be adjusted. This response ensures that only properly assembled ribosomes reach the cytoplasm for protein synthesis.
Implications for Cellular Health:
Coordinating ribosome biogenesis with the DNA replication stress response is essential for the well-being of the cell. This coordination ensures that the cellular machinery for protein synthesis remains robust and functional, even in the face of challenges to DNA replication. By doing so, cells can maintain their proteostasis and respond effectively to environmental and intrinsic stresses.

Miscellaneous Ribosome-Related Mechanisms

Ribosome Interactions with Viruses

Viruses, as obligate intracellular parasites, heavily rely on the host cellular machinery for their replication and propagation. One of the most critical components they interact with is the ribosome, responsible for protein synthesis. The nature of these interactions varies, but in eukaryotic cells, viruses have evolved numerous strategies to manipulate ribosomal functions to their advantage.

Role in Error Check and Repair:
When viruses interact with ribosomes, there's potential for errors in the translation of both viral and host mRNAs. Eukaryotic cells, in their bid to maintain homeostasis, constantly monitor these interactions to detect and rectify any anomalies. This ensures that the translation machinery remains functional, even in the presence of viral components.
Viral mRNAs and Ribosome Recruitment:
Many viruses have specialized sequences or structures in their mRNAs that allow for preferential recruitment of ribosomes. This ensures that viral proteins are synthesized efficiently, often at the expense of host proteins.
Ribosome Shunting:
Some viruses can induce a mechanism called ribosome shunting, where ribosomes bypass certain regions of mRNAs. This allows for the selective translation of specific viral proteins essential for the viral lifecycle.
Viral Inhibition of Host Translation:
Many viruses employ strategies to shut down host translation while ensuring that their mRNAs are efficiently translated. This can involve cleavage of translation initiation factors or modification of ribosomal components.
Ribosomal Frameshifting:
Certain viruses use a mechanism called ribosomal frameshifting to produce different proteins from a single mRNA. This involves the ribosome shifting to a different reading frame during translation, leading to the synthesis of an entirely different protein.
Ribosome Degradation:
To divert resources towards viral replication, some viruses induce degradation of host ribosomes. This not only conserves cellular resources but also reduces the translation of host defense proteins.
Interactions with Ribosomal Proteins:
Several viruses directly interact with ribosomal proteins, either to facilitate their replication or to inhibit specific host responses.
Implications for Cellular Health:
The interaction between viruses and ribosomes has significant implications for cellular health. By hijacking the ribosomal machinery, viruses can divert cellular resources, inhibit host defense mechanisms, and promote their replication. Understanding these interactions is crucial for the development of antiviral therapies and strategies to boost cellular defenses against viral infections.

Post-translation Quality Control Extant in Both

Recognition and Addressing of Misfolded Proteins

Ensuring the proper folding of nascent polypeptides is crucial to cellular health, as misfolded proteins can lead to aggregation, cellular dysfunction, and diseases. Various mechanisms and molecular players are engaged in assisting the correct folding of these proteins and handling those that become misfolded.

Ribosome-Associated Chaperones: These are protein complexes that associate directly with the ribosome, aiding in the folding of nascent polypeptides as they emerge from the ribosome.
NAC (Nascent chain-associated complex): It interacts with nascent polypeptides during their synthesis and may play roles in directing them towards proper folding pathways or to the appropriate cellular compartments.
RAC (Ribosome-associated complex): Comprising of the proteins Ssb and Zuo1 in yeast, this complex facilitates the folding of emerging polypeptides and can recruit other chaperones if needed.
Molecular Chaperones: These proteins facilitate the folding of nascent polypeptides and prevent them from aggregating.
Hsp70: It binds to nascent polypeptides, aiding in their folding and preventing aggregation. It also helps in refolding misfolded proteins.
Hsp90: Often working with co-chaperones, it stabilizes and assists in the correct folding of specific target proteins.
Proteostasis Network: This complex network of proteins ensures cellular protein homeostasis. It involves chaperones, the ubiquitin-proteasome system, and autophagy pathways.
Ubiquitin-Proteasome System: Misfolded proteins can be tagged with ubiquitin molecules for degradation by the proteasome.
Autophagy: Cellular mechanism that engulfs and degrades larger aggregates of misfolded proteins, which are too big for the proteasome to handle.
Endoplasmic Reticulum (ER) Quality Control: The ER has its system to ensure the proper folding of proteins meant for secretion or membrane insertion.
ER Chaperones: Such as BiP (a type of Hsp70) and calnexin, assist in protein folding within the ER.
ER-associated degradation (ERAD): Misfolded proteins in the ER can be retrotranslocated to the cytosol and then degraded by the ubiquitin-proteasome system.
Chaperone-Mediated Refolding: Some chaperones, like Hsp104 in yeast, can actively disaggregate and refold misfolded protein aggregates, restoring their functionality.

Intracellular Feedback Mechanisms

To maintain protein homeostasis, cells have feedback mechanisms that sense protein misfolding and induce responses to address it.
Heat Shock Response: In response to elevated temperatures or other stressors that can cause protein misfolding, cells upregulate the expression of heat shock proteins (Hsps) to help refold or degrade the affected proteins.
Unfolded Protein Response (UPR): Specifically sensing misfolded proteins in the ER, UPR triggers a cellular response that reduces protein translation, upregulates ER chaperones, and can initiate apoptosis if the stress is too severe.

Through these interconnected pathways and mechanisms, cells vigilantly maintain the health and functionality of their proteome, ensuring cellular efficiency and viability.

Ribosome-Associated Chaperones

Proper folding of nascent polypeptides as they emerge from ribosomes is crucial for ensuring functional proteins. Ribosome-associated chaperones play a key role in this process by assisting in the folding of these emerging polypeptides, preventing their aggregation, and directing them to the appropriate cellular compartments or pathways. These chaperones effectively couple translation with the initial steps of protein folding.

NAC (Nascent chain-associated complex): NAC is a heterodimeric complex composed of α and β subunits. It binds to nascent polypeptides during their synthesis, possibly shielding them from aggregation. NAC might also play roles in directing nascent chains toward the appropriate cellular compartments like the endoplasmic reticulum or mitochondria.
RAC (Ribosome-associated complex); In yeast, it's mainly composed of the proteins Ssb (an Hsp70 family member) and Zuo1. In higher eukaryotes, analogous systems exist. RAC facilitates the folding of emerging polypeptides. Ssb binds directly to the nascent chains, helping to prevent aggregation and facilitating folding. Zuo1, acting in concert with Ssb, can recruit other chaperones and co-chaperones if needed to assist in the folding process.
Trigger Factor (TF): TF is a ribosome-associated chaperone found in bacteria. TF binds to the ribosome near the polypeptide exit tunnel. As nascent polypeptides emerge, TF can bind to them, aiding in their initial folding steps. It acts as a holding chaperone, giving the nascent chain an opportunity to fold correctly before other interactions or aggregations can occur.
Hsp70 Systems: Comprises the main chaperone protein (like DnaK in bacteria or Ssb in yeast) and its co-chaperones. Hsp70 can bind to nascent polypeptides, aiding in their folding. In eukaryotes, the ribosome-associated Hsp70 (like Ssb) works with the RAC complex for this purpose.
Interplay with the SRP (Signal Recognition Particle): SRP is a ribonucleoprotein complex. While not a chaperone, SRP plays a role in recognizing and targeting nascent polypeptides with specific signal sequences to the endoplasmic reticulum for translocation. It can act in concert with or sometimes in competition with NAC, underscoring the complex interplay of these systems during co-translational protein targeting and folding.
These ribosome-associated chaperones ensure that as soon as a polypeptide starts emerging from the ribosome, it receives assistance in its folding process. This early intervention is vital, as it reduces the chances of misfolding or aggregation, promoting cellular health and efficiency.
Interactions with Molecular Chaperones: Chaperones like Hsp70 and Hsp90 help nascent polypeptides fold correctly.
Ribosome-Associated Translation Pausing: Mechanisms that introduce pauses during translation, which can influence the folding of proteins or set off downstream quality control processes.

Monitoring and Adjusting Translation

Ribosome Function Monitoring

Ensuring the accuracy and fidelity of protein synthesis is paramount for the health and function of the cell. Translation is a highly regulated process, and several mechanisms and molecular players are involved in monitoring ribosome function during translation. These mechanisms ensure that ribosomes synthesize proteins accurately, and any errors or problematic situations are quickly addressed.

Proofreading Mechanisms:
tRNA Selection: During the codon-anticodon recognition process, there are multiple kinetic checkpoints. Only the correct tRNA (carrying the appropriate amino acid) that matches the mRNA codon is accepted, while incorrect tRNAs are ejected.
Elongation Factor Tu (EF-Tu in bacteria): In bacteria, EF-Tu delivers aminoacyl-tRNAs to the ribosome. It plays a crucial role in discriminating between correctly and incorrectly charged tRNAs, ensuring that the correct amino acid is added to the growing polypeptide chain.

Ribosome Quality Control (RQC):
Stalled Ribosome Sensing: Certain sequences or secondary structures in mRNAs, or the absence of the appropriate tRNA, can cause ribosomes to stall. Cells have mechanisms to recognize these stalled ribosomes.
RQC Complex: In eukaryotes, when a ribosome stalls during translation, the RQC complex is recruited. It ubiquitinates the nascent polypeptide, marking it for degradation, and ensures the stalled ribosome is recycled.
tmRNA System (in bacteria): In bacteria, when a ribosome stalls due to a lack of a stop codon, transfer-messenger RNA (tmRNA) is used. tmRNA has properties of both tRNA and mRNA. It acts to add a tag to the C-terminus of the nascent polypeptide, marking it for proteolysis, and it also releases the stalled ribosome.

No-Go and Non-Stop Decay Pathways (in eukaryotes):
No-Go Decay: This targets mRNAs that cause ribosomes to stall, for example, due to strong secondary structures. The mRNA is endonucleolytically cleaved near the stall site and subsequently degraded.
Non-Stop Decay: Targets mRNAs that lack stop codons. The ribosome translates until it reaches the poly-A tail, and this triggers the mRNA's degradation.

Peptide Release Factors:
Release Factors: Proteins like RF1 and RF2 in bacteria, or eRF1 in eukaryotes, recognize stop codons and promote the release of the newly synthesized polypeptide from the ribosome. They ensure accurate termination of translation.
Ribosome Rescue Mechanisms: In both bacteria and eukaryotes, specific protein factors can recognize stalled ribosomes and promote their dissociation, freeing them to participate in new rounds of translation.
These intricate and overlapping mechanisms underscore the importance of accurate protein synthesis. By maintaining the fidelity of translation, cells prevent the accumulation of non-functional or potentially harmful proteins, thus promoting cellular health and homeostasis.

tRNA Modifications and Translation Fidelity

Transfer RNAs (tRNAs) play a central role in translation by delivering the correct amino acids to the ribosome in response to the codons in the messenger RNA (mRNA). To enhance their function and ensure translation fidelity, tRNAs undergo various post-transcriptional modifications. These modifications influence tRNA structure, stability, and interactions, thereby playing a vital role in the accuracy of protein synthesis.

Types of tRNA Modifications:
Methylations: Addition of methyl groups to specific bases or the ribose backbone.
Isomerizations: Conversion of one base to another, like inosine formation from adenine.
Thiolations: Addition of thiol (sulfur) groups.
Pseudouridylations: Conversion of uridine to pseudouridine.

Roles of tRNA Modifications in Translation Fidelity:
Codon-Anticodon Interactions: Modifications, especially in the anticodon loop of tRNAs, can enhance or influence the specificity of codon-anticodon base pairing. For example, inosine modifications can allow for wobble base pairing, increasing the flexibility of codon recognition. Pseudouridines in the anticodon loop can improve tRNA's conformational flexibility, ensuring optimal codon-anticodon interactions.
tRNA Stability and Structure: Modifications can stabilize the tRNA's tertiary structure, allowing it to be correctly recognized and bound by both the ribosome and aminoacyl-tRNA synthetases.
For instance, thiolations often provide protection against nucleases, extending the tRNA's lifespan in the cell.
Aminoacylation: The attachment of the correct amino acid to its corresponding tRNA by aminoacyl-tRNA synthetases is essential for translation fidelity. Modifications can influence this process, ensuring that the right amino acid is linked to the right tRNA.Some modifications can act as identity elements, helping aminoacyl-tRNA synthetases distinguish between similar tRNAs.
Ribosome Interactions: Modifications in certain regions of the tRNA can influence its interaction with the ribosome, ensuring proper positioning and movement of the tRNA within the ribosome during translation.
Preventing Frame Shifting: Accurate reading of mRNA codons is vital. Certain tRNA modifications can prevent the ribosome from slipping on the mRNA, which would lead to frame-shift mutations and erroneous protein synthesis.
Response to Cellular Stresses: Under specific stress conditions, such as nutrient starvation or extreme temperatures, unique tRNA modifications can occur. These modifications can adjust the translation process to the prevailing conditions, ensuring that essential proteins are synthesized even under stress.

Post-transcriptional modifications in tRNA molecules play a multifaceted role in ensuring translation fidelity. By influencing tRNA structure, interactions, and functionality, these modifications contribute to the accurate synthesis of proteins, maintaining cellular health and function.

mRNA 3' UTR and Translation Regulation

The 3' untranslated region (3' UTR) of messenger RNAs (mRNAs) is a segment that comes after the coding sequence but is not translated into protein. While it doesn't code for amino acids, the 3' UTR plays a pivotal role in post-transcriptional regulation of gene expression. It can influence mRNA stability, localization, and, importantly, translation efficiency. The 3' UTR often contains binding sites for various regulatory molecules that modulate these processes.

Roles of the 3' UTR in Translation Regulation:
MicroRNA (miRNA) Binding: miRNAs are small non-coding RNAs that can bind to complementary sequences in the 3' UTRs of target mRNAs. Upon binding, miRNAs typically reduce translation efficiency, either by inhibiting the initiation step of translation or by causing mRNA degradation. Different miRNAs can target a wide range of mRNAs, providing a broad mechanism for downregulating protein synthesis.
RNA Binding Proteins (RBPs): The 3' UTR contains binding sites for various RBPs. Depending on the specific RBP, binding can either inhibit or enhance translation. For instance, some RBPs might block the recruitment of the ribosome, while others can protect mRNA from degradation, thereby promoting translation. RBPs can also influence mRNA localization, ensuring translation occurs at specific cellular sites.
Circularization of mRNA: The 3' UTR can interact with the 5' UTR through proteins like poly(A) binding protein (PABP) and the eukaryotic translation initiation factor 4G (eIF4G).
This circularization can enhance translation by facilitating ribosome recycling, allowing multiple rounds of translation from the same mRNA molecule.
Alternative Polyadenylation: Many genes have multiple polyadenylation sites, leading to mRNAs with varying 3' UTR lengths. Depending on which site is used, different regulatory elements (like miRNA binding sites or RBP sites) can be included or excluded from the 3' UTR, influencing the mRNA's translational fate.
mRNA Stability and Decay: Certain elements in the 3' UTR can mark the mRNA for rapid degradation, reducing its translation. Conversely, stabilizing elements can increase an mRNA's half-life, leading to enhanced protein production.
Response to Cellular Signals: The 3' UTR can contain elements that respond to specific cellular conditions or signals. For example, in response to specific growth factors, proteins might bind to the 3' UTR and activate translation. Conversely, in response to stress signals, other proteins or miRNAs might bind and inhibit translation.
Subcellular Localization: Elements in the 3' UTR can direct mRNAs to specific cellular locations, such as synapses in neurons or the leading edge of migrating cells. Localized translation allows for precise spatial control of protein production. In essence, the 3' UTR serves as a regulatory hub, integrating multiple signals and modulating translation accordingly. This ensures that the right proteins are produced in the right amounts, at the right time, and in the right place within the cell.

Ribosome-Associated Translation Pausing

The process of translation, while often efficient and swift, isn't always continuous. At times, ribosomes pause at certain points on the mRNA during translation. Such pauses, far from being glitches, play vital roles in ensuring proper protein synthesis and function. Certain sequences in the mRNA can fold into stable secondary structures, such as hairpins, that temporarily impede ribosome progression until they're resolved. Infrequent codons, which correspond to low-abundance tRNAs, can cause the ribosome to slow down or pause, waiting for the appropriate tRNA to arrive. Specific sequences or motifs in the nascent polypeptide can cause pauses, especially if they interact with the ribosomal exit tunnel. Factors like the Signal Recognition Particle (SRP) or certain chaperones can bind to the emerging polypeptide or the ribosome itself, causing a pause in translation. By pausing translation, the ribosome allows time for the already synthesized portion of the protein to begin folding correctly. This can be especially important for large proteins or those with complex tertiary structures. Proteins destined for membranes often have hydrophobic sequences. Pausing allows the Signal Recognition Particle (SRP) to recognize and bind these sequences, ensuring the ribosome targets the endoplasmic reticulum (in eukaryotes) or the plasma membrane (in bacteria) for co-translational insertion. For proteins that are part of multi-subunit complexes, pausing can allow time for other subunits or cofactors to associate with the nascent chain, aiding in the correct assembly of the complex. Extended pauses, especially those caused by problematic mRNA sequences or damaged ribosomes, can be signals for quality control mechanisms. These mechanisms can then rescue the ribosome and degrade the faulty mRNA or nascent protein. Pausing can provide the necessary temporal window for enzymes to carry out modifications on the nascent polypeptide, such as phosphorylation or glycosylation. Ribosome-associated translation pausing is a strategic mechanism to ensure the fidelity and efficiency of protein synthesis. By coordinating the rate of translation with other cellular processes, pausing plays an indispensable role in the life of a protein, from its synthesis to its final function.

References

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3. Cvetesic, N., Perona, J., & Gruic‐Sovulj, I. (2012) Kinetic Partitioning in Class I Aminoacyl-tRNA Synthetases:The Journal of Biological Chemistry, 287, 25381 - 25394. Link.  
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5. Kumar Kuncha, S., Kruparani, S.P., & Sankaranarayanan, R. (2019). Chiral checkpoints during protein biosynthesis. J Biol Chem, 294(45):16535-16548. Link. (Discussion on the role of chiral checkpoints in protein biosynthesis.)
6. Kozak M. (2005). Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene, 361:13-37. Link.
7. Quality Control Mechanisms During Ribosome Maturation. Trends Cell Biol, 23(5):242-250. Link. (Description or summary of the content is not provided.)
8. Lund E, Dahlberg JE. (1998). Proofreading and aminoacylation of tRNAs before export from the nucleus. Science, 282(5396):2082-2085. Link. (Description or summary of the content is not provided.)
9. Kimura, S., & Waldor, M.K. (2019). The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA. Proc Natl Acad Sci U S A, 116(4):1394-1403. Link. (Discussion on the role of the RNA degradosome in tRNA quality control and its involvement in the clearance of hypomodified tRNA.)
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O ribossomo: Uma Fábrica Fundamental da Vida

O ribossomo é uma “fábrica” que produz todas as proteínas essenciais para a vida. Os ribossomos são fundamentais para a sobrevivência das células e da vida, sendo impossível imaginar células vivas sem elas, e não se conhece formas de vida sem elas. Cada ribossomo, forma e polimeriza aminoácidos para se tornarem cadeias completas de polímeros que se dobram para tornar formas finais de proteínas.

Os ribossomos são enormes em comparação com moléculas simples, contendo cerca de um milhão e quatrocentos mil átomos cada um. As células do figado do ser humano podem conter dezenas de milhares de ribossomos do seu citoplasma celular.

Ribossomos são verdadeiras fábricas digitais, muito mais competentes e confiáveis ​​do que impressoras 3D modernas. O ribossomo usa vinte aminoácidos, que possuem formas regulares e repetidas, como os tijolos de Lego. É interessante notar que 19 dos aminoácidos usados ​​nas células são homoquirais, ou seja, canhotos, fora da serina. Isso é essencial para polimerizar proteinas funcionais. Não há proteínas se não utilizar os 19 aminoácidos todos, 100% canhotos.

Uma mistura de aminoácidos heteroquirais não produz proteínas funcionais. E na terra prebiótica não havia como fazer essa seleção. As principais enzimas responsáveis ​​por garantir a produção de aminoácidos canhotos nas vias metabólicas celulares são: Racemases de aminoácidos, desidrogenases, Aminotransferases (transaminases), e Ligases e sintetases. Elas não existiam na terra prebiótica. 

A ação combinada dessas enzimas, altamente específicas para isômeros canhotos, garante que os aminoácidos produzidos e utilizados no metabolismo celular sejam predominantemente canhotos. Esta homoquiralidade é essencial para o correto dobramento e função das proteínas em sistemas biológicos. A falta de mecanismos prebióticos para selecionar os aminoácidos, só isso por si só, torna a origem da vida por meios não guiados, elatórios, impóssiveis.

The origin of the ribosome is considered a significant problem in understanding the origin of life because the ribosome is an incredibly complex molecular machine that is essential for all living organisms, yet its emergence through evolutionary mechanisms is difficult to explain.

The ribosome is a large, intricate structure composed of dozens of different RNA and protein components that work together to translate the genetic code into functional proteins. The ribosome is present in all three domains of life (Archaea, Bacteria, and Eukarya), suggesting it arose very early in the history of life.

The main challenges in explaining the origin of the ribosome through evolutionary mechanisms are:

1. Complexity: The ribosome is an extremely complex structure, with multiple interdependent components that must all be present and properly assembled for it to function. The probability of all these components arising and assembling by chance through random evolutionary processes is incredibly low.

2. Chicken-and-egg problem: The ribosome requires both RNA and proteins to function, yet the synthesis of proteins is dependent on the ribosome itself. This creates a chicken-and-egg problem – which came first, the ribosome or the proteins it produces?

3. Lack of intermediate forms: There is a lack of clear evolutionary intermediates or "stepping stones" that could demonstrate how the ribosome gradually arose through a series of small, incremental changes. The ribosome seems to appear in a largely complete, functional form.

4. Irreducible complexity: The ribosome is considered an example of "irreducible complexity" – the idea that certain biological systems are so complex that they cannot be produced by slight, successive modifications, as Darwin's theory of evolution by natural selection would predict.

These challenges have led some researchers to propose alternative, non-evolutionary explanations for the origin of the ribosome, such as intelligent design or the possibility that the ribosome may have arisen through a separate, non-Darwinian process that is not yet fully understood.

However, many evolutionary biologists continue to search for possible scenarios and mechanisms that could account for the origin of the ribosome through gradual, step-wise evolutionary processes. This remains an active area of research and debate in the field of origin of life studies.