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.     

Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues

The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA [Wagner, T., Cramer, F., & Sprinzl, M. (1982) Biochemistry 21, 1521-1529]. This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site. In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited. The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA. If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.     

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).     

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.     

Binding of [14C]tuberactinomycin O, an antibiotic closely related to viomycin, to the bacterial ribosome.

The binding of [¹⁴C]tuberactinomycin O, an antibiotic closely related to viomycin, to $\text{E. coli}$ ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA_F (initiation) and inhibition of translocation of peptidyl-tRNA.     

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.     

Single-molecule study of viomycin's inhibition mechanism on ribosome translocation

Viomycin belongs to the tuberactinomycin family of antibiotics against tuberculosis. However, its inhibition mechanism remains elusive. Although it is clear that viomycin inhibits the ribosome intersubunit ratcheting, there are contradictory reports about whether the antibiotic viomycin stabilizes the tRNA hybrid or classical state. By using a single-molecule FRET method to directly observe the tRNA dynamics relative to ribosomal protein L27, we have found that viomycin trapped the hybrid state within certain ribosome subgroups but did not significantly suppress the tRNA dynamics. The persistent fluctuation of tRNA implied that tRNA motions were decoupled from the ribosome intersubunit ratcheting. Viomycin also promoted peptidyl-tRNA fluctuation in the posttranslocation complex, implying that, in addition to acylated P-site tRNA, the decoding center also played an important role of ribosome locking after translocation. Therefore, viomycin inhibits translocation by trapping the hybrid state in the pretranslocation complex and disturbing the stability of posttranslocation complex. Our results imply that ribosome translocation is possibly a synergistic process of multiple decoupled local dynamics.     

Induction of translational errors (misreading) by tuberactinomycins and capreomycins

The well characterized translocation inhibitor viomycin (=tuberactinomycin B) promotes translational errors (misreading) in an in vitro system from Escherichia coli. It strongly stimulates both the binding of noncognate Tyr-tRNA to poly(U)-programmed ribosomes and the subsequent synthesis of acPhe(Tyr)_n-tRNA ($\text{n}\text{?20}$). The closely related antibiotics capreomycin and tuberactinomycins A,N and O also inhibit translocation and induce misreading.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

Peptidyl-CCA deacylation on the ribosome promoted by induced fit and the O3'-hydroxyl group of A76 of the unacylated A-site tRNA

The last step in ribosome-catalyzed protein synthesis is the hydrolytic release of the newly formed polypeptide from the P-site bound tRNA. Hydrolysis of the ester link of the peptidyl-tRNA is stimulated normally by the binding of release factors (RFs). However, an unacylated tRNA or just CCA binding to the ribosomal A site can also stimulate deacylation under some nonphysiological conditions. Although the sequence of events is well described by biochemical studies, the structural basis of the mechanism underlying this process is not well understood. Two new structures of the large ribosomal subunit of Haloarcula marismortui complexed with a peptidyl-tRNA analog in the P site and two oligonucleotide mimics of unacylated tRNA, CCA and CA, in the A site show that the binding of either CA or CCA induces a very similar conformational change in the peptidyl-transferase center as induced by aminoacyl-CCA. However, only CCA positions a water molecule appropriately to attack the carbonyl carbon of the peptidyl-tRNA and stabilizes the proper orientation of the ester link for hydrolysis. We, thus, conclude that both the ability of the O3′-hydroxyl group of the A-site A76 to position the water and the A-site CCA induced conformational change of the PTC are critical for the catalysis of the deacylation of the peptidyl-tRNA by CCA, and perhaps, an analogous mechanism is used by RFs.     

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.     

Inhibition of polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides

Macrolides, lincosamides and type B synergimycins are powerful inhibitors of protein synthesis in vivo, but many of them were found to be inactive in vitro. In the present work, we confirm that virginiamycin S (a type B synergimycin) and erythromycin (a 14-membered macrolide) have no effect on poly(U)-directed poly(Phe) synthesis. However, the amino-acid polymerization reactions directed by poly(U,G), poly(U,C), poly(A,G) and poly(A,C) were increasingly inhibited (20-50%) by both antibiotics. The action of these inhibitors proved to be template-dependent and favored by the incorporation of proline and of basic amino acids into peptides. Under these conditions, virginiamycin S and erythromycin markedly stimulated a release of peptidyl-tRNA from the ribosomes. In the poly(A,C) model system, these antibiotics produced a 50% inhibition of amino-acid incorporation into total peptides, a 70% release of ribosome-bound peptidyl-tRNA, and a 95% repression of the synthesis of long peptide chains. The production of equivalent effects at saturating concentrations of these antibiotics in the four model systems examined is suggestive of a similarity in their mode of action. Our results indicate that 14-membered macrolides and type B synergimycins can act on ribosomes during the whole elongation process. The functional block produced by both antibiotics is usually reversible, but may result in a premature release of peptidyl-tRNA when the stability of ribosomal complexes is lowered by the incorporation of basic amino acids.     

The role of translocation in ribosomal accuracy. Translocation rates for cognate and noncognate aminoacyl- and peptidyl-tRNAs on Escherichia coli ribosomes

The ribosomal translocation, as measured in vitro by peptide formation on poly(U)-programmed Escherichia coli ribosomes in the presence of ternary complex, deacylated tRNA or N-acetyl-Phe-tRNA, and elongation factor G, is the rate-limiting step of protein synthesis. Elongation factor G stimulates the spontaneous translocation by a factor of about 500. N-Acetyl-Phe-Phe-tRNA(Phe E. coli) is translocated with a rate constant of 1-2 s-1 at 25 degrees C. Translocation of N-acetyl-Phe-Phe-tRNA(Phe yeast) and N-acetyl-Phe-Leu-tRNA(Leu E. coli) under identical conditions proceeds with a rate by about a factor of 2 and 10, respectively, more slowly. The translocation rate, therefore, is influenced by the nature of the tRNAs in the A-site. We can show, furthermore, that also the tRNA in the P-site, and presumably in the E-site as well, influences the rate of translocation. Reduced rates of translocation of noncognate peptidyl-tRNAs are accompanied by preferential dissociation of these tRNAs at the beginning of the translation of a mRNA.     

The antibiotic viomycin traps the ribosome in an intermediate state of translocation

During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.     

Inhibition by Thiopeptin of Bacterial Protein Synthesis

Thiopeptin, a sulfur-containing antibiotic, was found to inhibit protein synthesis in a bacterial ribosomal system. The pretreatment of ribosomal subunits with the antibiotic revealed that thiopeptin may act on the 50 S ribosomal subunit. The elongation of peptide chain on the ribosome is more profoundly blocked by the antibiotic than the initiation of protein synthesis. It was demonstrated that thiopeptin inhibits elongation factor (EF)-Tu-dependent GTP hydrolysis and binding of aminoacyl-tRNA to the ribosome. The peptidyl transferase-catalyzed puromycin reaction is not significantly affected by the antibiotic. Thiopeptin inhibits EF-G-associated GTPase reaction, and translocation of peptidyl-tRNA and mRNA from the acceptor site to the donor site. Protein synthesis in ribosomal systems, obtained from rat liver and rabbit reticulocytes are insensitive to the antibiotic.     

Eukaryotic elongation factor 2 can bind to the synthetic oligoribonucleotide that mimics sarcin/ricin domain of rat 28S ribosomal RNA

Eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to P site by binding to the ribosome. In this work, the complex formation of rat liver eEF2 with a synthetic oligoribonucleotide (SRD RNA) that mimics sarcin/ricin domain of rat 28S ribosomal RNA is invested in vitro. Purified eEF2 can specifically bind SRD RNA to form a stable complex. tRNA competes with SRD RNA in binding to eEF2 in a less extent. Pretreatment of eEF2 with GDP or ADP-ribosylation of eEF2 by diphtheria toxin can obviously reduce the ability of eEF2 to form the complex with the synthetic oligoribonucleotide. These results indicate that eEF2 is likely to bind directly to the sarcin/ricin domain of 28S ribosomal RNA in the process of protein synthesis.     

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.     

Universally conserved interactions between the ribosome and the anticodon stem-loop of A site tRNA important for translocation

The iterative movement of the tRNA-mRNA complex through the ribosome is a hallmark of the elongation phase of protein synthesis. We used synthetic anticodon stem-loop analogs (ASL) of tRNA(Phe) to systematically identify ribose 2'-hydroxyl groups that are essential for binding and translocation from the ribosomal A site. Our results show that 2'-hydroxyl groups at positions 33, 35, and 36 in the A site ASL are important for translocation. Consistent with the view that the molecular basis of translocation may be similar in all organisms, the 2'-hydroxyl groups at positions 35 and 36 in the ASL interact with universally conserved bases G530 and A1493, respectively, in 16S rRNA. Furthermore, these interactions are also essential for the decoding process, indicating a functional relationship between decoding and translocation.     

The mode of action of alpha sarcin and a novel assay of the puromycin reaction.

A new technique was developed for measuring the amount of peptidyl-tRNA in a protein-synthesizing system in vitro. By this technique the course of the puromycin reaction may be followed and the modes of action of various inhibitors of protein synthesis readily determined. We conclude that the polypeptide alpha sarcin inhibits the binding of aminoacyl-tRNA into the ribosomal 'A' site, that sparsomycin inhibits the peptidyl transferase reaction and that cycloheximide may block translocation.