Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner.

Ribosomes complexed with synthetic mRNA and peptidyl-tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide-releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non-hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + P(i) was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.     

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex.

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets. We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.     

Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides.

Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.     

Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli

Release factors RF1 and RF2 recognize stop codons present at the A-site of the ribosome and activate hydrolysis of peptidyl-tRNA to release the peptide chain. Interactions with RF3, a ribosome-dependent GTPase, then initiate a series of reactions that accelerate the dissociation of RF1 or RF2 and their recycling between ribosomes. Two regions of Escherichia coli RF1 and RF2 were identified previously as involved in stop codon recognition and peptidyl-tRNA hydrolysis. We show here that removing the N-terminal domain of RF1 or RF2 or exchanging this domain between the two factors does not affect RF specificity but has different effects on the activity of RF1 and RF2: truncated RF1 remains highly active and able to support rapid cell growth, whereas cells with truncated RF2 grow only poorly. Transplanting a loop of 13 amino acid residues from RF2 to RF1 switches the stop codon specificity. The interaction of the truncated factors with RF3 on the ribosome is defective: they fail to stimulate guanine nucleotide exchange on RF3, recycling is not stimulated by RF3, and nucleotide-free RF3 fails to stabilize the binding of RF1 or RF2 to the ribosome. However, the N-terminal domain seems not to be required for the expulsion of RF1 or RF2 by RF3:GTP.     

Timing of GTP binding and hydrolysis by translation termination factor RF3

Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.     

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.     

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.     

RF3:GTP promotes rapid dissociation of the class 1 termination factor

Translation termination is promoted by class 1 and class 2 release factors in all domains of life. While the role of the bacterial class 1 factors, RF1 and RF2, in translation termination is well understood, the precise contribution of the bacterial class 2 release factor, RF3, to this process remains less clear. Here, we use a combination of binding assays and pre-steady state kinetics to provide a kinetic and thermodynamic framework for understanding the role of the translational GTPase RF3 in bacterial translation termination. First, we find that GDP and GTP have similar affinities for RF3 and that, on average, the t_1/2 for nucleotide dissociation from the protein is 1–2 min. We further show that RF3:GDPNP, but not RF3:GDP, tightly associates with the ribosome pre- and post-termination complexes. Finally, we use stopped-flow fluorescence to demonstrate that RF3:GTP enhances RF1 dissociation rates by over 500-fold, providing the first direct observation of this step. Importantly, catalytically inactive variants of RF1 are not rapidly dissociated from the ribosome by RF3:GTP, arguing that a rotated state of the ribosome must be sampled for this step to efficiently occur. Together, these data define a more precise role for RF3 in translation termination and provide insights into the function of this family of translational GTPases.     

Binding of reticulocyte elongation factor 1 to ribosomes and nucleic acids.

The present study has examined the requirements for the binding of rabbit reticulocyte elongation factor 1 (EF-1) to ribosomes under different assay conditions. When a centrifugation procedure was used to separate the ribosome EF-1 complex, the binding of EF-1 to ribosomes required GTP and Phe-tRNA, but not poly(U). The results suggested that undr these conditions a ternary complex, EF-1 . GTP . aminoacyl-tRNA, is necessary for the formation of a ribosome . EF-1 complex. However, when gel filtration was used to isolate the ribosome . EF-1 complex, only template and tRNA were required. These studie emphasize the fact that the procedure used to isolate the ribosome . EF-1 complex determines the requirements for stable complex formation. EF-1 can also interact with nucleic acids such as 28 S and 18 S rRNA, messenger RNA and DNA. In contrast to the binding to ribosomes, EF-1 binding to nucleic acids requires only Mg2+.     

Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.

Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.     

Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3.

A complete translation system has been assembled from pure initiation, elongation and termination factors as well as pure aminoacyl-tRNA synthetases. In this system, ribosomes perform repeated rounds of translation of short synthetic mRNAs which allows the time per translational round (the recycling time) to be measured. The system has been used to study the influence of release factor RF3 and of ribosome recycling factor RRF on the rate of recycling of ribosomes. In the absence of both RF3 and RRF, the recycling time is approximately 40 s. This time is reduced to approximately 30 s by the addition of RF3 alone and to approximately 15 s by the addition of RRF alone. When both RF3 and RRF are added to the translation system, the recycling time drops to <6 s. Release factor RF3 is seen to promote RF1 cycling between different ribosomes. The action of RRF is shown to depend on the concentration of elongation factor-G. Even in the presence of RRF, ribosomes do not leave the mRNA after termination, but translate the same mRNA several times. This shows that RRF does not actively eject mRNA from the terminating ribosome. It is proposed that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalysed consecutively by RF3 and RRF, which are slow in the absence of these factors.     

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.     

Reduced turnover of the elongation factor EF-1 X ribosome complex after treatment with the protein synthesis inhibitor II from barley seeds

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [14C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 X GTP X aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.     

Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

The mechanism of the protein-synthesis elongation cycle in eukaryotes. Effect of ricin on the ribosomal interaction with elongation factors

The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pre-translocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1-catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 X GTP X aminoacyl-tRNA X ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1- and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.     

Novel roles for classical factors at the interface between translation termination and initiation.

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.     

Reduced ribosomal binding of eukaryotic elongation factor 2 following ADP-ribosylation. Difference in binding selectivity between polyribosomes and reconstituted monoribosomes

The biological activity of elongation factor 2 (EF-2) following NAD+ - and diphtheria-toxin-dependent ADP-ribosylation was studied (i) in translation experiments using the reticulocyte lysate system and (ii) in ribosomal binding experiments using either reconstituted empty rat liver ribosomes or programmed reticulocyte polysomes. Treatment of the lysates with toxin and NAD+ at a NAD+/ribosome ratio of 4 resulted in a 90% inhibition of the amino acid incorporation rate. The inhibition was overcome by the addition of native EF-2. At this level of inhibition more than 90% of the EF-2 present in the lysates was ADP-ribosylated and the total ribosome association of EF-2 was reduced by approx. 50%. All of the remaining unmodified factor molecules were associated with the ribosomes, whereas only about 3% of the ribosylated factor was ribosome-associated. The nucleotide requirement for the binding of EF-2 to empty reconstituted rat liver ribosomes and programmed reticulocyte polysomes was studied together with the stability of the resulting EF-2 X ribosome complexes using purified 125I-labelled rat liver EF-2. With both types of ribosomes, the complex formation was strictly nucleotide-dependent. Stable, high-affinity complexes were formed in the presence of the non-hydrolysable GTP analogue guanosine 5'-(beta, gamma-methylene)triphosphate (GuoPP[CH2]P). In contrast to the reconstituted ribosomes, GTP stimulated the formation of high-affinity complexes in the presence of polysomes, albeit at a lower efficiency than GuoPP[CH2]P. The formation of high-affinity complexes was restricted to polysomes in the pretranslocation phase of the elongation cycle. Low-affinity post-translocation complexes, demonstrable after fixation, were formed in the presence of GTP, GuoPP[CH2]P and GDP. In polysomes, these complexes involved a different population of particles than did the high-affinity complexes. In the binding experiments using reconstituted or programmed ribosomes, the pretranslocation binding of EF-2 observed in the presence of GuoPP[CH2]P was reduced by approx. 50% after ADP-ribosylation, whereas the post-translocation binding in the presence of GDP was unaltered. The data indicate that the inhibition of translocation caused by diphtheria toxin and NAD+ is mediated through a reduced affinity of the ADP-ribosylated EF-2 for binding to ribosomes in the pretranslocation state.     

The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2-GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.