Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp > Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.     

The Identification of the Determinants of the Cyclic, Sequential Binding of Elongation Factors Tu and G to the Ribosome

Experiments dedicated to gaining an understanding of the mechanism underlying the orderly, sequential association of elongation factor Tu (EF-Tu) and elongation factor G (EF-G) with the ribosome during protein synthesis were undertaken. The binding of one EF is always followed by the binding of the other, despite the two sharing the same—or a largely overlapping—site and despite the two having isosteric structures. Aminoacyl-tRNA, peptidyl-tRNA, and deacylated-tRNA were bound in various combinations to the A-site, P-site, or E-site of ribosomes, and their effect on conformation in the peptidyl transferase center, the GTPase-associated center, and the sarcin/ricin domain (SRD) was determined. In addition, the effect of the ribosome complexes on sensitivity to the ribotoxins sarcin and pokeweed antiviral protein and on the binding of EF-G•GTP were assessed. The results support the following conclusions: the EF-Tu ternary complex binds to the A-site whenever it is vacant and the P-site has peptidyl-tRNA; and association of the EF-Tu ternary complex is prevented, simply by steric hindrance, when the A-site is occupied by peptidyl-tRNA. On the other hand, the affinity of the ribosome for EF-G•GTP is increased when peptidyl-tRNA is in the A-site, and the increase is the result of a conformational change in the SRD. We propose that peptidyl-tRNA in the A-site is an effector that initiates a series of changes in tertiary interactions between nucleotides in the peptidyl transferase center, the SRD, and the GTPase-associated center of 23S rRNA; and that the signal, transmitted through a transduction pathway, informs the ribosome of the position of peptidyl-tRNA and leads to a conformational change in the SRD that favors binding of EF-G.     

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.     

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.     

Function of the ribosomal E-site: a mutagenesis study

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.     

The translation initiation functions of IF2: targets for thiostrepton inhibition

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.     

Properties of elongation factor G: its interaction with the ribosomal peptidyl-site

EF-G bound to poly(U)·ribosomes prevents enzymatic or nonenzymatic binding of charged tRNA not only to the A-site but also to the P-site. In turn, charged tRNA bound either to the P- or A-site prevents formation of EF-G·GMPPCP·ribosome complex. Ribosomes carrying newly synthetized peptidyl-tRNA in pretranslocative state are also unable to form stable complexes with EF-G. The functional implications of these observations are discussed and it is suggested that tRNA plays a regulatory role in the interaction of EF-G with ribosomes during the cyclic process of elongation.     

Blasticidin S inhibits mammalian translation and enhances production of protein encoded by nonsense mRNA

Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3′CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1’s accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.     

Functional sites of interaction between release factor RF1 and the ribosome

Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor.     

Perturbation of the tRNA tertiary core differentially affects specific steps of the elongation cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.     

The kinetics of ribosomal peptidyl transfer revisited

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.     

Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA

Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.     

Single-molecule fluorescence measurements of ribosomal translocation dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.     

Photoaffinity modification of Escherichia coli ribosomes near the tRNA-binding centers by tRNAPhe derivatives carrying arylazido groups on guanosine residues

Photoreactive derivatives of tRNAPhe (E. coli) bearing arylazido groups scattered statistically over guanosine residues (azido-tRNA) were applied for affinity labelling of E. coli ribosomes in elongation factor-dependent or factor-free model systems mimicking different steps of elongation. It is shown that UV-irradiation of the corresponding complexes of ribosomes with tRNA derivatives results in labelling of both subunits, the 30S one is labelled preferentially. In all experiments only ribosomal proteins were labelled. Comparison of the sets of proteins labelled by tRNA derivatives in different states at P-site allowed us to draw important conclusions concerning the influence of peptidyl moiety and of the occupancy of the A-site with aminoacyl- or peptidyl-tRNA on the arrangement of tRNA located at the P-site. Three of the 30S proteins--S11, S13 S19--are labelled with tRNA derivatives located at P-site in all states.     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.     

Ribosomes in a Stacked Array: ELUCIDATION OF THE STEP IN TRANSLATION ELONGATION AT WHICH THEY ARE STALLED DURING S-ADENOSYL-l-METHIONINE-INDUCED TRANSLATION ARREST OF CGS1 mRNA*

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.     

Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs

The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.     

Functional importance of the 3'-terminal adenosine of tRNA in ribosomal translation

The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA(Val) A76G. Val-tRNA(CCG) does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA(CCG) represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA(CCG) to the A- or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-CCG-tRNA from the P- to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G(76) from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.     

Action of erythromycin and virginiamycin S on polypeptide synthesis in cell-free systems

Erythromycin (a 14-membered macrolide) and virginiamycin S (a type B synergimycin) block protein biosynthesis in bacteria, but are virtually inactive on poly(U)-directed poly(Phe) synthesis. We have recently shown, however, that these antibiotics inhibit the in vitro polypeptide synthesis directed by synthetic copolymers: this effect is analyzed further in the present work. We were unable to find any consistent alteration produced by these antibiotics on coupled and uncoupled EF-G- and EF-Tu-dependent GTPases, on the EF-Tu-directed binding of aminoacyl-tRNA to ribosomes, and on the EF-G- and GTP-mediated translocation of peptidyl-tRNA bound to poly(U,C).ribosome complexes. With these complexes, the peptidyl transfer reaction, as measured by peptidylpuromycin synthesis, was 10-30% inhibited by virginiamycin S and erythromycin. A direct relationship between the virginiamycin S- and erythromycin-promoted inhibition of poly(A,C)-directed polypeptide synthesis, on the one hand, and the EF-G concentration and the rate of the polymerization reaction, on the other hand, was observed, in agreement with a postulated reversible inhibitor action of these antibiotics. The increased inhibitory activity, which was observed during the first 4-6 rounds of elongation, in the presence of virginiamycin S or erythromycin, was suggestive of a specific action of these antibiotics on the correct positioning of peptidyl-tRNA at the P site. The marked stimulation of premature release of peptidyl-tRNA from poly(A,C).ribosome complexes can be referred to an altered interaction of the C-terminal aminoacyl residue of the growing peptidyl chain with the ribosome. We conclude that the action of virginiamycin S and erythromycin entails a template-dependent alteration of the interaction of peptidyl-tRNA with the donor site of peptidyltransferase, which may lead to a transient functional block of the ribosome and in some instances to a premature release of peptidyl-tRNA and termination of the elongation process.