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.     

Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.     

Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G

After peptide release by a class-1 release factor, the ribosomal subunits must be recycled back to initiation. We have demonstrated that the distance between a strong Shine-Dalgarno (SD) sequence and a codon in the P site is crucial for the binding stability of the deacylated tRNA in the P site of the posttermination ribosome and the in-frame maintenance of its mRNA. We show that the elongation factor EF-G and the ribosomal recycling factor RRF split the ribosome into subunits in the absence of initiation factor 3 (IF3) by a mechanism that requires both GTP and GTP hydrolysis. Taking into account that EF-G in the GTP form and RRF bind with positive cooperativity to the free 50S subunit but with negative cooperativity to the 70S ribosome, we suggest a mechanism for ribosome recycling that specifies distinct roles for EF-G, RRF, and IF3.     

Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.     

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.     

Release of ribosome-bound ribosome recycling factor by elongation factor G

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.     

Sequence of steps in ribosome recycling as defined by kinetic analysis

After termination of protein synthesis in bacteria, ribosomes are recycled from posttermination complexes by the combined action of elongation factor G (EF-G), ribosome recycling factor (RRF), and initiation factor 3 (IF3). The functions of the factors and the sequence in which ribosomal subunits, tRNA, and mRNA are released from posttermination complexes are unclear and, in part, controversial. Here, we study the reaction by rapid kinetics monitoring fluorescence. We show that RRF and EF-G with GTP, but not with GDPNP, promote the dissociation of 50S subunits from the posttermination complex without involving translocation or a translocation-like event. IF3 does not affect subunit dissociation but prevents reassociation, thereby masking the dissociating effect of EF-G-RRF under certain experimental conditions. IF3 is required for the subsequent ejection of tRNA and mRNA from the small subunit. The latter step is slower than subunit dissociation and constitutes the rate-limiting step of ribosome recycling.     

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 recycling factor disassembles the post-termination ribosomal complex independent of the ribosomal translocase activity of elongation factor G

Ribosome recycling factor (RRF) disassembles post-termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex independently of the translocation activity of EF-G.     

Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5′-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-Å (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S·EF-G·guanosine 5′-[(βγ)-imido]triphosphate·RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5′-[(βγ)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.     

The ribosome-recycling step: consensus or controversy?

Ribosome recycling, the last step in translation, is now accepted as an essential process for prokaryotes. In 2005, three laboratories showed that ribosome-recycling factor (RRF) and elongation factor G (EF-G) cause dissociation of ribosomes into subunits, solving the long-standing problem of how this essential step of translation occurs. However, there remains ongoing controversy regarding the other actions of RRF and EF-G during ribosome recycling. We propose that the available data are consistent with the notion that RRF and EF-G not only split ribosomes into subunits but also participate directly in the release of deacylated tRNA and mRNA for the next round of translation.     

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.     

Isolation and characterization of an inhibitor of ribosome-dependent GTP hydrolysis by elongation factor G

Two inhibitors of ribosome-dependent GTP hydrolysis by elongation factor (EF)G were found in the ribosome wash of Escherichia coli strain B. One of these inhibitors was purified to homogeneity and characterized. The isolated inhibitor was found to consist of two polypeptide subunits with apparent molecular masses of 23 kDa and 10 kDa. Inhibition of EF-G GTPase could not be overcome by increasing amounts of the elongation factor or high concentrations of GTP, but was reversed by large amounts of ribosomes. The effect of the inhibitor was reduced by increasing concentrations of either 30S or 50S ribosomal subunits. EF-G-dependent GTPase of 50S ribosomal subunits was not affected by the inhibitor. These findings clearly show that the inhibitor interferes with the modulation of EF-G GTPase activity by the interactions between 30S and 50S ribosomal subunits. Under conditions, where 30S CsCl core particles are able to associate with 50S subunits and to stimulate EF-G GTPase, the effect of the inhibitor was considerably reduced when intact 30S ribosomal subunits were substituted by 30S CsCl core particles. This finding indicates that 30S CsCl split proteins are important for the action of the inhibitor and that the inhibitor does not affect the EF-G GTPase merely by interfering with the association of ribosomal subunits. Furthermore, poly(U)-dependent poly(phenylalanine) synthesis was considerably less sensitive to the inhibitor than EF-G GTPase. When ribosomes were preincubated with poly(U) and Phe-tRNA(Phe), poly(phenylalanine) synthesis was considerably less affected by the inhibitor, whereas EF-G GTPase was still sensitive.     

Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies

Ribosome recycling, the disassembly of the posttermination complex after each round of protein synthesis, is an essential step in mRNA translation, but its mechanism has remained obscure. In eubacteria, recycling is catalyzed by RRF (ribosome recycling factor) and EF-G (elongation factor G). By using cryo-electron microscopy, we have obtained two density maps, one of the RRF bound posttermination complex and one of the 50S subunit bound with both EF-G and RRF. Comparing the two maps, we found domain I of RRF to be in the same orientation, while domain II in the EF-G-containing 50S subunit is extensively rotated (approximately 60 degrees) compared to its orientation in the 70S complex. Mapping the 50S conformation of RRF onto the 70S posttermination complex suggests that it can disrupt the intersubunit bridges B2a and B3, and thus effect a separation of the two subunits. These observations provide the structural basis for the mechanism by which the posttermination complex is split into subunits by the joint action of RRF and EF-G.     

Mechanism of the ribosome-dependent uncoupled GTPase reaction catalyzed by polypeptide chain elongation factor G.

At low NH4-+ concentrations, 50S ribosomal subunits from E. coli were fully active in the absence of 30S ribosomal subunits, in forming a complex with the polypeptide chain elongation factor G (EF-G) and guanine nucleotide (ternary complex formation), and also in supporting EF-G dependent hydrolysis of GTP (uncoupled GTPase reaction). However, both activities were markedly inhibited on increasing the concentration of the monovalent cation, and at 160 mM NH4-+, the optimal concentration for polypeptide synthesis in a cell-free system, almost no activity was observed with 50S ribosomes alone. It was found that the inhibitory effect of NH4-+ was reversed by addition of 30S subunits. Thus, at 160 mM NH4-+, only 70S ribosomes were active in supporting the above two EF-G dependent reactions, whereas at 20 mM NH4-+, 50S ribosomes were almost as active as 70S ribosomes. Kinetic studies on inhibition by NH4-+ of the formation of 50S ribosome-EF-G-guanine nucleotide complex, indicated that the inhibition was due to reduction in the number of active 50S ribosomes which were capable of interacting with EF-G and GTP at higher concentrations of NH4-+. The inhibitory effects of NH4-+ on ternary complex formation and the uncoupled GTPase reaction were markedly influenced by temperature, and were much greater at 0 degrees than at 30 degrees. A conformational change of 50S subunits through association with 30S subunits is suggested.     

Mechanism of recycling of post-termination ribosomal complexes in eubacteria: A new role of initiation factor 3

Ribosome recycling is a process which dissociates the post-termination complexes (post-TC) consisting of mRNA-bound ribosomes harbouring deacylated tRNA(s). Ribosome recycling factor (RRF), and elongation factor G (EFG) participate in this crucial process to free the ribosomal subunits for a new round of translation. We discuss the overall pathway of ribosome recycling in eubacteria with especial reference to the important role of the initiation factor 3 (IF3) in this process. Depending on the step(s) at which IF3 function is implicated, three models have been proposed. In model 1, RRF and EFG dissociate the post-TCs into the 50S and 30S subunits, mRNAand tRNA(s). In this model, IF3, which binds to the 30S subunit, merely keeps the dissociated subunits apart by its anti-association activity. In model 2, RRF and EFG separate the 50S subunit from the post-TC. IF3 then dissociates the remaining complex of mRNA, tRNA and the 30S subunit, and keeps the ribosomal subunits apart from each other. However, in model 3, both the genetic and biochemical evidence support a more active role for IF3 even at the step of dissociation of the post-TC by RRF and EFG into the 50S and 30S subunits.     

Recycling of eukaryotic posttermination ribosomal complexes

After translational termination, mRNA and P site deacylated tRNA remain associated with ribosomes in posttermination complexes (post-TCs), which must therefore be recycled by releasing mRNA and deacylated tRNA and by dissociating ribosomes into subunits. Recycling of bacterial post-TCs requires elongation factor EF-G and a ribosome recycling factor RRF. Eukaryotes do not encode a RRF homolog, and their mechanism of ribosomal recycling is unknown. We investigated eukaryotic recycling using post-TCs assembled on a model mRNA encoding a tetrapeptide followed by a UAA stop codon and report that initiation factors eIF3, eIF1, eIF1A, and eIF3j, a loosely associated subunit of eIF3, can promote recycling of eukaryotic post-TCs. eIF3 is the principal factor that promotes splitting of posttermination ribosomes into 60S subunits and tRNA- and mRNA-bound 40S subunits. Its activity is enhanced by eIFs 3j, 1, and 1A. eIF1 also mediates release of P site tRNA, whereas eIF3j ensures subsequent dissociation of mRNA.     

Chaperone properties of bacterial elongation factor EF-G and initiation factor IF2

Elongation factor G(EF-G) and initiation factor 2 (IF2) are involved in the translocation of ribosomes on mRNA and in the binding of initiator tRNA to the 30 S ribosomal subunit, respectively. Here we report that the Escherichia coli EF-G and IF2 interact with unfolded and denatured proteins, as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-G and IF2 promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They prevent the aggregation of citrate synthase under heat shock conditions, and they form stable complexes with unfolded proteins such as reduced carboxymethyl alpha-lactalbumin. Furthermore, the EF-G and IF2-dependent renaturations of citrate synthase are stimulated by GTP, and the GTPase activity of EF-G and IF2 is stimulated by the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. The concentrations at which these chaperone-like functions occur are lower than the cellular concentrations of EF-G and IF2. These results suggest that EF-G and IF2, in addition to their role in translation, might be implicated in protein folding and protection from stress.     

Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits

After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.     

An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3?

An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-[35S]Met-tRNAfMet to ribosomes. In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-[35S]Met-tRNAfMET/poly(A,U,G)/ribosome complexes. Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet. Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3. The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor. Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits.