Tests of the ribosome editor hypothesis

Summary Peptidyl-tRNA dissociates from the ribosomes of Escherichia coli during protein biosynthesis. The ribosome editor hypothesis states that incorrect peptidyl-tRNAs dissociate preferentially. Editing would therefore prevent the completion of proteins containing misincorporated amino acids. We have isolated a mutant strain of E. coli that dissociates some peptidyl-tRNAs at a fivefold lower rate than its parent strain, and that synthesizes significantly more erroneous complete proteins. This strain is also partially resistant to the antibiotic erythromycin, which in wildtype E. coli stimulates the dissociation of peptidyl-tRNA from ribosomes. The data suggest that in this mutant all peptidyl-tRNAs are bound to the ribosome more tightly than normally during protein synthesis. Because of the inverse correlation between the accuracy of synthesis of complete proteins and the rate of dissociation of peptidyl-tRNA from the ribosome, we propose that the mutant contains a defective ribosomal editor.     

Computer simulation of ribosome editing

A stochastic model of protein synthesis was modified by including the process of dissociating peptidyl-tRNA from ribosomes. To simulate ribosome editing, the probability of dissociation was assumed to be high if the peptidyl-tRNA was erroneous; that is, if it resulted from transfer of a peptide to an aminoacyl-tRNA that was inappropriate relative to the mRNA codon. The effects of amino acid starvation on protein synthesis were simulated both by increasing the probability of such erring at and by reducing the conditional probability of elongation at “hungry” codons, those whose correct amino acid was in short supply. These probabilities were varied systematically to simulate tryptophan limitation during synthesis of coat protein from bacteriophage MS2.Significant reduction, during starvation, in the synthesis of complete coat protein required large reductions in the probability of elongation at hungry codons but only small increases in the probability of erring. Enhanced dissociation of peptidyl-tRNA during starvation, followed rapidly by dissociation of ribosomes from mRNA, led to reductions in mean polysome size, a result that had been interpreted by others as due to some effect of starvation on the initiation of protein synthesis.Results from experiments by Goldman (1982) on the cell-free synthesis of MS2 coat protein during tryptophan starvation could be mimicked in detail by the computer simulations. A simple competition between correct and erroneous amino acids was sufficient to explain the tryptophan dependence of complete coat protein and internal peptide syntheses. Values for the Michaelis constants were derived from the computer simulations.     

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.     

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation. Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site. Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6. Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6. Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids. To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation. Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation. The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction. Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml). A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation. We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells.     

The mechanism of action of macrolides,lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome

The macrolide-lincosamide-streptogramin B class (MLS) of antibiotics contains structurally different but functionally similar drugs, that all bind to the 50S ribosomal subunit. It has been suggested that these compounds block the path by which nascent peptides exit the ribosome. We have studied the mechanisms of action of four macrolides (erythromycin, josamycin, spiramycin and telithromycin), one lincosamide (clindamycin) and one streptogramin B (pristinamycin IA). All these MLS drugs cause dissociation of peptidyl-tRNA from the ribosome. Josamycin, spiramycin and clindamycin, that extend to the peptidyl transferase center, cause dissociation of peptidyl-tRNAs containing two, three or four amino acid residues. Erythromycin, which does not reach the peptidyl transferase center, induces dissociation of peptidyl-tRNAs containing six, seven or eight amino acid residues. Pristinamycin IA causes dissociation of peptidyl-tRNAs with six amino acid residues and telithromycin allows polymerisation of nine or ten amino acid residues before peptidyl-tRNA dissociates. Our data, in combination with previous structural information, suggest a common mode of action for all MLS antibiotics, which is modulated by the space available between the peptidyl transferase center and the drug.     

tRNAs: Cellular barcodes for amino acids

The role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl-tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis.     

Genetically encoded but nonpolypeptide prolyl-tRNA functions in the A site for SecM-mediated ribosomal stall

The arrest sequence, FXXXXWIXXXXGIRAGP, of E. coli SecM interacts with the ribosomal exit tunnel, thereby interfering with translation elongation. Here, we studied this elongation arrest in vitro using purified translation components. While a simplest scenario would be that elongation is arrested beyond Pro166, the last arrest-essential amino acid, and that the Pro166 codon is positioned at the P site of the ribosomal peptidyl transferase center (PTC), our toeprint analyses revealed that the ribosome actually stalls when the Pro166 codon is positioned at the A site. Northern hybridization identification of the polypeptide bound tRNA and mass determination showed that the last amino acid of the arrested peptidyl-tRNA is Gly165, which is only inefficiently transferred to Pro166. Also, puromycin does not effectively release the arrested peptidyl-tRNA under the conditions of A site occupancy by Pro166-tRNA. These results reveal that secM-encoded Pro166-tRNA functions as a nonpolypeptide element in fulfilling SecM's role as a secretion monitor.     

Inhibition of peptide chain termination by antibodies specific for ribosomal proteins

The effects of antibodies specific for the Escherichia coli 30 S and 50 S ribosomal proteins have been determined for in vitro peptide chain termination and two partial reactions, the codon-directed binding of E. coli release factor to the ribosome and peptidyl-tRNA hydrolysis with RF². Antibodies to ribosomal proteins L7 and L12 inhibit the initial binding of RF to the ribosome, and as a result, the subsequent peptidyl-tRNA hydrolysis. The kinetics of ribosomal inactivation for in vitro termination by anti-L7/L12 indicate that Fab fragments bind to three ribosome sites, and suggest that each of three copies of L7/L12 is involved in the binding of RF to the ribosome. When 70 S ribosome substrates are pretreated with anti-L11 and anti-L16 RF-dependent peptidyl-tRNA, hydrolysis is partially inhibited but the interaction of RF with the ribosome is not affected. The inactivation of in vitro termination by a mixture of anti-L11 and anti-L16 is not co-operative. Pretreatment of the 30 S ribosomal subunit (but not 70 S ribosomal substrate) with antibodies to the 30 S proteins, S9 and S11, results in strong inhibition of codon-directed hydrolysis of peptidyl-tRNA. While these antibodies inhibit ribosome subunit association, a requirement for peptide chain termination, and thereby may inhibit the in vitro termination reactions indirectly, the codon-directed binding of RF is markedly more affected than peptidyl-tRNA hydrolysis by anti-S9 and anti-S11. Antibody to S2 and anti-S3 exhibit a similar but less marked differential effect on the partial reactions of in vitro termination under the same conditions. When dissociated ribosomes are pretreated with anti-L11, in vitro termination is completely inhibited and both codon-directed binding of RF and peptidyl-tRNA hydrolysis are affected. L11 may, therefore, be at or near the interface between the ribosome subunits and like S9 and S11 not completely accessible to antibody in 70 S ribosomes. Pretreatment of dissociated ribosomes with antibodies to a number of other ribosomal proteins (L2, L4, L6, L14, L15, L17, L18, L20, L23, L26, L27) results in partial inhibition of all termination reactions although these antibodies have no effect on termination when incubated with 70 S ribosome substrates. The antibodies probably affect in vitro termination indirectly as a result of either preventing correct ribosome subunit association, or preventing correct positioning of the fMet-tRNA at the ribosome P site.     

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn⁻ phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn⁻ phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn⁻ phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.     

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.     

Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein

High-level expression of non-functional model proteins, derived from elongation factor EF-Tu by the deletion of an essential domain, greatly inhibits the growth of Escherichia coli partly deficient in peptidyl-tRNA hydrolase. High-level expression in wild-type cells has little effect on growth. The inhibitory effect is therefore presumably due to the sequestration of essential tRNA species, partly in the form of free peptidyl-tRNA. The growth inhibitory effect can be modulated by changing the last sense codon in the genes encoding the model proteins. Thus, replacement of Ser by Lys or His at this position increases growth inhibition. The effects of 11 changes studied are related to the rates of accumulation previously observed of the corresponding families of peptidyl-tRNA. Two non-exclusive hypotheses are proposed to account for these observations: first, the last sense codon of mRNA is a prefered site of peptidyl-tRNA drop-off in cells, due to the slow rate of translation termination compared with sense codon translation; secondly, the relatively long pause of the ribosome at the stop codon (of the order of 1 s), results in significant temporary sequestration on the ribosome of the tRNA cognate to the last sense codon.     

Kinetics of macrolide action: the josamycin and erythromycin cases

Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of full-length proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacterio-toxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyl-tRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.     

The suppression of defective translation by ppGpp and its role in the stringent response.

Amino acid starvation is shown to decrease the fidelity of translation in E. coli. When proteins are analyzed by two-dimensional gel electrophoresis, missense errors are detected as an unusual heterogeneity in their isoelectric points, while premature termination of protein synthesis can be recognized by a decreased relative rate of synthesis of higher molecular weight proteins and by the the accumulation of a complex group of new small polypeptides. The types of translational errors observed are amino acid-specific. For example, starvation of a rel- strain for histidine produces severe isoelectric point heterogeneity with little evidence of premature termination, while starvation for leucine has little effect on the isoelectric points, but produces a drastic decrease in the average molecular weight of the newly synthesized protein. These differences suggest codon-specific errors in reading the genetic code. In these rel- cells, the effect of amino acid starvation on the rates of synthesis of complete individual proteins is both protein- and amino acid-specific. For example, ribosomal protein L7/12, which lacks histidine, is made at a higher level during histidine starvation than during isoleucine or leucine starvation. This suggests that in rel- cells, the modulation of gene expression caused by the lack of a particular amino acid is, at least in part, a function of the abundance of that amino acid in particular proteins-that is, the response of rel- cells to starvation is consistent with the theory that the inhibition of protein synthesis and the accompanying increase in error frequency both result from low levels of the correct substrate. In marked contrast, virtually no starvation-induced translational errors are detected in a rel+ strain, and the response is not amino acid-specific. Varoius data strongly imply that in this rel+ strain, essentially all the changes caused by starvation are due to the accumulation of ppGpp, which independently reduces protein synthesis, thereby suppressing all the direct effects of amino acid limitation seen in rel- strains (where ppGpp does not accumulate upon starvation). A model is presented which describes how ppGpp might suppress the direct effects of starvation and avoid the loss of translational fidelity. In addition, the direct and specific effects of ppGpp on gene expression are examined independently of amino acid starvation.     

Drop-off-reinitiation triggered by EF-G-driven mistranslocation and its alleviation by EF-P

In ribosomal translation, peptidyl transfer occurs between P-site peptidyl-tRNA and A-site aminoacyl-tRNA, followed by translocation of the resulting P-site deacylated-tRNA and A-site peptidyl-tRNA to E and P site, respectively, mediated by EF-G. Here, we report that mistranslocation of P-site peptidyl-tRNA and A-site aminoacyl-tRNA toward E and A site occurs when high concentration of EF-G triggers the migration of two tRNAs prior to completion of peptidyl transfer. Consecutive incorporation of less reactive amino acids, such as Pro and d-Ala, makes peptidyl transfer inefficient and thus induces the mistranslocation event. Consequently, the E-site peptidyl-tRNA drops off from ribosome to give a truncated peptide lacking the C-terminal region. The P-site aminoacyl-tRNA allows for reinitiation of translation upon accommodation of a new aminoacyl-tRNA at A site, leading to synthesis of a truncated peptide lacking the N-terminal region, which we call the ‘reinitiated peptide’. We also revealed that such a drop-off-reinitiation event can be alleviated by EF-P that promotes peptidyl transfer of Pro. Moreover, this event takes place both in vitro and in cell, showing that reinitiated peptides during protein synthesis could be accumulated in this pathway in cells.     

Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest

SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain outside the ribosome affects the stability of translation arrest. To clarify this issue, we performed in vitro translation assays with HaloTag proteins fused to the C-terminal fragment of E. coli SecM containing the arrest sequence or the full-length SecM. We showed that the translation of HaloTag proteins, which are fused to the fragment, is not effectively arrested, whereas the translation of HaloTag protein fused to full-length SecM is arrested efficiently. In addition, we observed that the nascent SecM chain outside the ribosome markedly stabilizes the translation arrest. These results indicate that changes in the nascent polypeptide chain outside the ribosome can affect the stability of translation arrest; the nascent SecM chain outside the ribosome stabilizes the translation arrest.     

The busiest of all ribosomal assistants: elongation factor Tu

During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, elongation, and termination. After a protein has been synthesized, the components of the translation apparatus are recycled. During each phase of translation, the ribosome collaborates with specific translation factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the elongation step is repeated a large number of times, corresponding to the number of amino acids constituting the protein of interest. In bacteria, elongation factor Tu plays a central role during the selection of the correct amino acids throughout the elongation phase of translation. Elongation factor Tu is the main subject of this review.     

Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center

The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role.     

Auxin-induced Changes in the Incorporation of H-Amino Acids into Soybean Ribosomal Proteins

Auxin-induced activation of 80S ribosomes and polyribosome formation in mature soybean (Glycine max var. Hawkeye) hypocotyl (R. L. Travis, J. M. Anderson, and J. L. Key. 1973. Plant Physiol. 52: 608-612) in the presence of a mixture of radioactive amino acids correlates with an increased specific radioactivity of at least three ribosomal proteins; the labeling of one of these increased severalfold above the control level. Results of experiments with 5-fluorouracil and cycloheximide indicated that the proteins in question were synthesized in response to auxin and became associated with pre-existing ribosomes. Ribosome dissociation experiments indicated that these proteins were associated with the 60S ribosome subunit.