Action of virginiamycin M on the stability of different ribosomal complexes to ultracentrifugation

It was previously shown that virginiamycin M produces in vivo an accumulation of pressure-sensitive (60 S) ribosomes, and in vitro an inactivation of the donor and acceptor sites of peptidyl transferase. The latter action, however, is expected to cause the accumulation in vivo of ribosome complexes carrying acylated tRNA species: such complexes are usually endowed with pressure resistance. However, present data indicate that poly(U).ribosome complexes carrying Phe-tRNA, Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at either the A or the P site become pressure-sensitive after exposure to virginiamycin M in vitro. It is known also that uncoupled EF-G GTPase is stimulated by P-site-bound unacylated tRNA, not by the acylated species. Our data show, however, a stimulation of EF-G GTPase, when ribosomal complexes carrying Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at the P site are incubated with virginiamycin M. The interpretation proposed to account for all these findings is that complexes carrying A- and P-site-bound aminoacyl-tRNA derivatives, which undergo a stable interaction with the peptidyl transferase, are endowed with ultracentrifugal stability, whereas complexes with unacylated tRNA (which does not interact with the enzyme) are pressure-sensitive. By inactivating the donor and acceptor sites of peptidyltransferase, virginiamycin M causes aminoacyl-tRNA.ribosome complexes to mimic tRNA.ribosome complexes in their pressure-lability and competence in EF-G GTPase stimulation. This interpretation is supported by the finding that the ribosome-promoted protection of aminoacyl-tRNA against spontaneous hydrolysis is suppressed by virginiamycin M.     

Transit of tRNA through the Escherichia coli ribosome. Cross-linking of the 3' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at the A, P, and E sites

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.     

The action of virginiamycin M on the acceptor, donor, and catalytic sites of peptidyltransferase

Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S). In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as well as after its removal. Virginiamycin M inhibited the binding of puromycin to ribosomes and reduced both the enzymatic and nonenzymatic binding of Phe-tRNA to the A site by inducing its release from the ribosomes (similar effects were observed with 50 S), whereas the antibiotic had no effect on the binding of unacylated tRNAPhe to the same site. Moreover, virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP (similar finding with 50 S). Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic. The overall conclusion is that both the acceptor and donor substrate binding sites of the peptidyltransferase, which interact with the aminoacyl moiety of tRNA, are permanently altered upon transient contact of ribosomes with virginiamycin M.     

New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome

Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3' adenosine of yeast tRNA(Phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-O-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA(Phe) and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center.     

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding

Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.     

Effect of spermine on the efficiency and fidelity of the codon-specific binding of tRNA to the ribosomes

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNA(Phe) and 5 for tRNA(Tyr). The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.     

An apparent conformational change in tRNA(Phe) that is associated with the peptidyl transferase reaction

Fluorescence techniques were used to detect changes in the conformation of tRNA(Phe) that may occur during the peptidyl transferase reaction in which the tRNA appears to move between binding sites on ribosomes. Such a conformational change may be a fundamental part of the translocation mechanism by which tRNA and mRNA are moved through ribosomes. E. coli tRNA(Phe) was specifically labeled on acp3U47 and s4U8 or at the D positions 16 and 20. The labeled tRNAs were bound to ribosomes as deacylated tRNA(Phe) or AcPhe-tRNA. Changes in fluorescence quantum yield and anisotropy were measured upon binding to the ribosomes and during the peptidyl transferase reaction. In one set of experiments non-radiative energy transfer was measured between a coumarin probe at position 16 or 20 and a fluorescein attached to acp3U47 on the same tRNA(Phe) molecule. The results indicate that the apparent distance between the probes increases during deacylation of AcPhe-tRNA as a result of peptide bond formation. All of the results are consistent with but in themselves do not conclusively establish that tRNA undergoes a conformational change as well as movement during the peptidyl transferase reaction.     

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.     

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.     

ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl‐tRNA hydrolysis in Escherichia coli

The ribosomes stalled at the end of non‐stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non‐productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans‐translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome‐tethered peptidyl‐tRNAs. ArfB is a paralogue of the release factors (RFs) and directly catalyses the peptidyl‐tRNA hydrolysis within NTCs. In contrast, the mechanism of the ArfA action had remained obscure beyond its ability to bind to the ribosome. Here, we characterized the ArfA pathway of NTC resolution in vitro and identified RF2 as a factor that cooperates with ArfA to hydrolyse peptidyl‐tRNAs located in the P‐site of the stalled ribosome. This reaction required the GGQ (Gly–Gly–Gln) hydrolysis motif, but not the SPF (Ser–Pro–Phe) codon–recognition sequence, of RF2 and was stimulated by tRNAs. From these results we suggest that ArfA binds to the vacant A‐site of the stalled ribosome with possible aid from association with a tRNA, and then recruits RF2, which hydrolyses peptidyl‐tRNA in a GGQ motif‐dependent but codon‐independent manner. In support of this model, the ArfA‐RF2 pathway did not act on the SecM‐arrested ribosome, which contains an aminoacyl‐tRNA in the A‐site.     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.     

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.     

Hygromycin A, a novel inhibitor of ribosomal peptidyltransferase

In cell-free systems from Escherichia coli, hygromycin A inhibits polypeptide synthesis directed by either poly(U) or phage R 17 RNA, and the reaction of puromycin with either natural peptidyl-tRNA, or AcPhe-tRNA, or the 3'-terminal fragment of AcLeu-tRNA (C-A-C-C-A-LeuAc). In contrast, the antibiotic does no inhibit the enzymatic binding of Phe-tRNA to ribosomes or the translocation of AcPhe-tRNA. It is concluded that hygromycin A is a specific inhibitor of the peptide bond formation step of protein synthesis. The action of hygromycin A on peptidyl transfer is similar to that of chloramphenicol, an antibiotic that shares some common structural features with hygromycin A. Both antibiotics inhibit the binding of C-A-C-C-A-Leu to the acceptor site of peptidyl transferase and stimulate that of C-A-C-C-A-LeuAc to the donor site of the enzyme. Moreover, hygromycin A blocks the binding of chloramphenicol to ribosomes, indicating that the binding sites of the antibiotics may be closely related. Hygromycin A is a more potent agent than chloramphenicol and binds quite strongly to ribosomes.     

Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster.

Scanning transmission electron microscopic images of transfer RNAs reveal the molecular dimensions and compact morphology of these small macromolecules in unprecedented detail. Selective labeling of a sulfhydryl group on 2-thiocytidine enzymatically inserted at position 75 at the 3' end of yeast tRNA(Phe) with an undecagold cluster permits identification of this specific tRNA site by dark field STEM. Imaging of a single nucleotide at a defined location on the tRNA molecule should make it possible to localize in situ tRNAs at the A, P, and E sites of the ribosomal peptidyl transferase center, and in complexes of tRNA with enzymes and elongation factors. In addition, this approach may be used for the highly specific topographical mapping of other RNAs and/or biological macromolecular complexes.     

Apparent association constants of tRNAs for the ribosomal A, P, and E sites.

Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.     

Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors

Alpha-sarcin cleaves one phosphodiester bond of 23S rRNA within 70S ribosomes or 50S subunits derived from E. coli. The resulting fragment was isolated and sequenced. The cleavage site was identified as being after G2661 and is located within a universally conserved dodecamer. Cleavage after G2661 specifically blocked the binding of both elongation factors, i.e. that of the ternary complex Phe-tRNA*EF-Tu*GMPPNP and of EF-G*GMPPNP, whereas all elongation-factor independent functions of the ribosome, such as association of the ribosomal subunits, tRNA binding to A and P sites, the accuracy of tRNA selection at both sites, the peptidyl transferase activity, and the EF-G independent, spontaneous translocation, were not affected at all. Control experiments with wheat germ ribosomes yielded an equivalent inhibition pattern. The data suggest that the universally conserved dodecamer containing the cleavage site G2661 is located at the presumably overlapping region of the binding sites of both elongation factors.     

Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.

The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more than a few angstroms during peptide bond formation, as judged by the movement of fluorescent probes attached to the phenylalanine amino group of phenylalanyl-tRNA. Other results demonstrate that an analogue of peptidyl-tRNA, deacylated tRNA, and puromycin can be bound simultaneously to the same ribosome, indicating that there are three physically distinct sites to which tRNA is bound during the reaction steps by which peptides are elongated. The results appear to be consistent with the displacement model of peptide elongation.     

A new simpler photoaffinity analogue of peptidyl tRNA

The synthesis of the n-hydroxysuccinimide ester of N-(2-nitro-4-azidophenyl)glycine (NAG) is described. This reacts with E. coli phe-tRNA(Phe) to yield the photoaffinity label NAG-Phe-tRNA(Phe). This peptidyl tRNA analogue binds correctly to the peptidyl site of the E. coli ribosome. The only significant covalent products found after irradiation of a peptidyl site bound NAG-Phe-tRNA(Phe)-70S-poly(U) complex are 50S proteins L11 and L18. After irradiation the complex can still bind [(3)H]Phe-tRNA to the amino acyl site and participate in peptide bond formation with the covalently attached NAG-Phe moiety. Alternatively, one can allow peptide bond formation to occur first, prior to photolysis. The reaction products are still L11 and L18. Hence, both of these two proteins appear to be centrally located at the peptidyl transferase center.     

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.     

Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.

A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.