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.     

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.     

Distinct functions of elongation factor G in ribosome recycling and translocation

Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.     

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.     

Coupling of GTP hydrolysis by elongation factor G to translocation and factor recycling on the ribosome

The translocation step of elongation entails the coordinated movement of tRNA and mRNA on the ribosome. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis, which affects both translocation and turnover of EF-G. Both reactions are much slower (50-100-fold) when GTP is replaced with non-hydrolyzable GTP analogues or GDP, indicating that the reaction rates are determined by conformational transitions induced by GTP hydrolysis. Compared to the rate of uncatalyzed, spontaneous translocation, ribosome binding of EF-G with any guanine nucleotide reduces the free energy of activation by about 18 kJ/mol, whereas GTP hydrolysis contributes another 10 kJ/mol. The acceleration by GTP hydrolysis is due to large decrease in activation enthalpy by about 30 kJ/mol, compared to the reaction with GTP analogues or GDP, whereas the activation entropy becomes unfavorable and is lowered by about 20 kJ/mol (37 degrees C). The data suggest that GTP hydrolysis induces, by a conformational change of EF-G, a rapid conformational rearrangement of the ribosome ("unlocking") which determines the rates of both tRNA-mRNA translocation and recycling of the factor.     

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.     

Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly

Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.     

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.     

Elongation factor G protects a nuclease-sensitive site of 23 S RNA within the ribosome

Elongation factor G is shown to protect the nuclease splitting off the 3′ -terminal 11 S fragment from the 23 S RNA within the ribosomal 50 S subparticle.     

Role of domains 4 and 5 in elongation factor G functions on the ribosome

Elongation factor G (EF-G) is a large, five domain GTPase that catalyses the translocation of the tRNAs on the bacterial ribosome at the expense of GTP. In the crystal structure of GDP-bound EF-G, domain 1 (G domain) makes direct contacts with domains 2 and 5, whereas domain 4 protrudes from the body of the molecule. Here, we show that the presence of both domains 4 and 5 is essential for tRNA translocation and for the turnover of the factor on the ribosome, but not for rapid single-round GTP hydrolysis by EF-G. Replacement of a highly conserved histidine residue at the tip of domain 4, His583, with lysine or arginine decreases the rate of tRNA translocation at least 100-fold, whereas the binding of the factor to the ribosome, GTP hydrolysis and P(i) release are not affected by the mutations. Various small deletions in the tip region of domain 4 decrease the translocation activity of EF-G even further, but do not block the turnover of the factor. Unlike native EF-G, the mutants of EF-G lacking domains 4/5 do not interact with the alpha-sarcin stem-loop of 23 S rRNA. These mutants are not released from the ribosome after GTP hydrolysis or translocation, indicating that the contact with, or a conformational change of, the alpha-sarcin stem-loop is required for EF-G release from the ribosome.     

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.     

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF‐G) in bacteria plays two distinct roles in different phases of the translation system. EF‐G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post‐termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF‐G are distributed over two different EF‐G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF‐G is also found in bacteria. Two EF‐G paralogues are found in the spirochaete Borrelia burgdorferi, EF‐G1 and EF‐G2. We demonstrate that EF‐G1 is a translocase, while EF‐G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF‐G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF‐3) is present in the reaction. These results indicate that two B. burgdorferi EF‐G paralogues are close relatives to mitochondrial EF‐G paralogues rather than the conventional bacterial EF‐G, in both their phylogenetic and biochemical features.     

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.     

Elongation factor G and protein S12 are the nearest neighbours in the Escherichia coli ribosome

It has been found that iodine oxidation of the complex of ribosomes with EF-G² results in the formation of a disulphide bond between the single exposed sulphydryl group of the factor and each of the ribosomal subunits. It is concluded that this EF-G region is localized on the ribosomal interface.Protein S12 is identified as the ribosomal component crosslinked with EF-G in the oxidation reaction. The possible role of the specific interaction of EF-G with protein S12 for the functioning of the factor as an activator of translocation is discussed.     

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.     

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 role of GTP in transient splitting of 70S ribosomes by RRF (ribosome recycling factor) and EF-G (elongation factor G)

Ribosome recycling factor (RRF), elongation factor G (EF-G) and GTP split 70S ribosomes into subunits. Here, we demonstrated that the splitting was transient and the exhaustion of GTP resulted in re-association of the split subunits into 70S ribosomes unless IF3 (initiation factor 3) was present. However, the splitting was observed with sucrose density gradient centrifugation (SDGC) without IF3 if RRF, EF-G and GTP were present in the SDGC buffer. The splitting of 70S ribosomes causes the decrease of light scattering by ribosomes. Kinetic constants obtained from the light scattering studies are sufficient to account for the splitting of 70S ribosomes by RRF and EF-G/GTP during the lag phase for activation of ribosomes for the log phase. As the amount of 70S ribosomes increased, more RRF, EF-G and GTP were necessary to split 70S ribosomes. In the presence of a physiological amount of polyamines, GTP and factors, even 0.6 μM 70S ribosomes (12 times higher than the 70S ribosomes for routine assay) were split. Spermidine (2 mM) completely inhibited anti-association activity of IF3, and the RRF/EF-G/GTP-dependent splitting of 70S ribosomes.