In vivo effect of inactivation of ribosome recycling factor - fate of ribosomes after unscheduled translation downstream of open reading frame

The post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Without RRF, the ribosome is not released from mRNA at the termination codon and reinitiates translation downstream. This is called unscheduled translation. Here, we show that at the non-permissive temperature of a temperature-sensitive RRF strain, RRF is lost quickly, and some ribosomes reach the 3' end of mRNA. However, instead of accumulating at the 3' end of mRNA, ribosomes are released as monosomes. Some ribosomes are transferred to transfer-messenger RNA from the 3' end of mRNA. The monosomes thus produced are able to translate synthetic homopolymer but not natural mRNA with leader and canonical initiation signal. The pellet containing ribosomes appears to be responsible for rapid but reversible inhibition of most but not all of protein synthesis in vivo closely followed by decrease of cellular RNA and DNA synthesis.     

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.     

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.     

Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli

Once the translating ribosomes reach a termination codon, the nascent polypeptide chain is released in a factor-dependent manner. However, the P-site-bound deacylated tRNA and the ribosomes themselves remain bound to the mRNA (post-termination complex). The ribosome recycling factor (RRF) plays a vital role in dissociating this complex. Here we show that the Mycobacterium tuberculosis RRF (MtuRRF) fails to rescue Escherichia coli LJ14, a strain temperature-sensitive for RRF (frr(ts)). More interestingly, co-expression of M.tuberculosis elongation factor G (MtuEFG) with MtuRRF rescues the frr(ts) strain of E.coli. The simultaneous expression of MtuEFG is also needed to cause an enhanced release of peptidyl-tRNAs in E.coli by MtuRRF. These observations provide the first genetic evidence for a functional interaction between RRF and EFG. Both the in vivo and in vitro analyses suggest that RRF does not distinguish between the translating and terminating ribosomes for their dissociation from mRNA. In addition, complementation of E.coli PEM100 (fusA(ts)) with MtuEFG suggests that the mechanism of RRF function is independent of the translocation activity of EFG.     

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.     

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.     

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.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

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.     

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.     

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine-Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.     

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.     

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.     

Crystal structure of the ribosome recycling factor bound to the ribosome

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.     

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.     

Ribosome recycling revisited

Ribosome recycling involves the coordinated action of the ribosome recycling factor (RRF), elongation factor EF-G and initiation factor IF3 to disassemble the post-termination complex, recycling the components for the next round of translation. The crystal structure of domain I of RRF (RRF-DI) in complex with the large ribosomal subunit from the eubacteria Deinococcus radiodurans at high resolution reveals the nature and details of the interactions between this protein factor and rRNA/protein components of the ribosome. Universally conserved arginine residues within the RRF-DI establish important interactions with nuleotides of the 23S rRNA, explaining why mutations at these positions abolish factor binding. Furthermore, in conjunction with cryo-EM reconstruction, the X-ray analysis provides a structural complement to the recent biochemical data, offering additional insight into the mechanism of ribosome recycling.     

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.     

Binding of ribosome recycling factor to ribosomes,comparison with tRNA

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.     

Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing

Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation. The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA. Directed hydroxyl radical probing of 16S and 23S rRNA from Fe(II) tethered to ten positions on the surface of E. coli RRF constrains it to a well-defined location in the subunit interface cavity. Surprisingly, the orientation of RRF in the ribosome differs markedly from any of those previously observed for tRNA, suggesting that structural mimicry does not necessarily reflect functional mimicry.