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.     

Kinetics and thermodynamics of RRF, EF-G, and thiostrepton interaction on the Escherichia coli ribosome

Ribosome recycling factor (RRF) and elongation factor-G (EF-G) are jointly essential for recycling bacterial ribosomes following termination of protein synthesis. Here we present equilibrium and rapid kinetic measurements permitting formulation of a minimal kinetic scheme that accounts quantitatively for RRF and EF-G interaction on the Escherichia coli ribosome. RRF and EF-G (a) each form a binary complex on binding to a bare ribosome which undergoes isomerization to a more stable complex, (b) form mixed ternary complexes on the ribosome in which the affinity for each factor is considerably lower than its affinity for binding to a bare ribosome, and (c) each bind to two sites per ribosome, with EF-G having considerably higher second-site affinity than RRF. Addition of EF-G to the ribosome-RRF complex induces rapid RRF dissociation, at a rate compatible with the rate of ribosome recycling in vivo, but added RRF does not increase the lability of ribosome-bound EF-G. Added thiostrepton slows the initial binding of EF-G, and prevents both formation of the more stable EF-G complex and EF-G-induced RRF dissociation. These findings are relevant for the mechanism of post-termination complex disassembly.     

Conformational changes in switch I of EF‐G drive its directional cycling on and off the ribosome

We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA–mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by ∼20 Å), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G•GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.     

Crosslinking of translation factor EF-G to proteins of the bacterial ribosome before and after translocation

Elongation factor G (EF-G) promotes the translocation of tRNA and mRNA in the central cavity of the ribosome following the addition of each amino acid residue to a growing polypeptide chain. tRNA/mRNA translocation is coupled to GTP hydrolysis, catalyzed by EF-G and activated by the ribosome. In this study we probed EF-G interactions with ribosomal proteins (r-proteins) of the bacterial ribosome, by using a combination of chemical crosslinking, immunoblotting and mass spectroscopy analyses. We identified three bacterial r-proteins (L7/L12, S12 and L6) crosslinked to specific residues of EF-G in three of its domains (G', 3 and 5, respectively). EF-G crosslinks to L7/L12 and S12 were indistinguishable when EF-G was trapped on the ribosome before or after tRNA/mRNA translocation had occurred, whereas a crosslink between EF-G and L6 formed with greater efficiency before translocation had occurred. EF-G crosslinked to L7/L12 was capable of catalyzing multiple rounds of GTP hydrolysis, whereas EF-G crosslinked to S12 was inactive in GTP hydrolysis. These results imply that during the GTP hydrolytic cycle EF-G must detach from S12 within the central cavity of the ribosome, while EF-G can remain associated with L7/L12 located on one of the peripheral stalks of the ribosome. This mechanism may ensure that a single GTP molecule is hydrolyzed for each tRNA/mRNA translocation event.     

Control of phosphate release from elongation factor G by ribosomal protein L7/12

Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.     

New insights into the enzymatic role of EF-G in ribosome recycling

During translation, elongation factor G (EF-G) plays a catalytic role in tRNA translocation and a facilitative role in ribosome recycling. By stabilizing the rotated ribosome and interacting with ribosome recycling factor (RRF), EF-G was hypothesized to induce the domain rotations of RRF, which subsequently performs the function of splitting the major intersubunit bridges and thus separates the ribosome into subunits for recycling. Here, with systematic mutagenesis, FRET analysis and cryo-EM single particle approach, we analyzed the interplay between EF-G/RRF and post termination complex (PoTC). Our data reveal that the two conserved loops (loop I and II) at the tip region of EF-G domain IV possess distinct roles in tRNA translocation and ribosome recycling. Specifically, loop II might be directly involved in disrupting the main intersubunit bridge B2a between helix 44 (h44 from the 30S subunit) and helix 69 (H69 from the 50S subunit) in PoTC. Therefore, our data suggest a new ribosome recycling mechanism which requires an active involvement of EF-G. In addition to supporting RRF, EF-G plays an enzymatic role in destabilizing B2a via its loop II.     

Brownian dynamics study of the association between the 70S ribosome and elongation factor G

Protein synthesis on the ribosome involves a number of external protein factors that bind at its functional sites. One key factor is the elongation factor G (EF-G) that facilitates the translocation of transfer RNAs between their binding sites, as well as advancement of the messenger RNA by one codon. The details of the EF-G/ribosome diffusional encounter and EF-G association pathway still remain unanswered. Here, we applied Brownian dynamics methodology to study bimolecular association in the bacterial EF-G/70S ribosome system. We estimated the EF-G association rate constants at 150 and 300 mM monovalent ionic strengths and obtained reasonable agreement with kinetic experiments. We have also elucidated the details of EF-G/ribosome association paths and found that positioning of the L11 protein of the large ribosomal subunit is likely crucial for EF-G entry to its binding site.     

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.     

Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.     

Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome

Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome.     

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.     

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.     

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.     

Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome

The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.     

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.     

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.     

Crystal structure of a mutant elongation factor G trapped with a GTP analogue

Elongation factor G (EF-G) is a G protein factor that catalyzes the translocation step in protein synthesis on the ribosome. Its GTP conformation in the absence of the ribosome is currently unknown. We present the structure of a mutant EF-G (T84A) in complex with the non-hydrolysable GTP analogue GDPNP. The crystal structure provides a first insight into conformational changes induced in EF-G by GTP. Comparison of this structure with that of EF-G in complex with GDP suggests that the GTP and GDP conformations in solution are very similar and that the major contribution to the active GTPase conformation, which is quite different, therefore comes from its interaction with the ribosome.     

How can elongation factors EF-G and EF-Tu discriminate the functional state of the ribosome using the same binding site?

Elongation factors EF-G and EF-Tu are structural homologues and share near-identical binding sites on the ribosome, which encompass the GTPase-associated centre (GAC) and the sarcin-ricin loop (SRL). The SRL is fixed structure in the ribosome and contacts elongation factors in the vicinity of their GTP-binding site. In contrast, the GAC is mobile and we hypothesize that it interacts with the alpha helix D of the EF-Tu G-domain in the same way as with the alpha helix A of the G'-domain of EF-G. The mutual locations of these helices and GTP-binding sites in the structures of EF-Tu and EF-G are different. Thus, the orientation of the GAC relative to the SRL determines whether EF-G or EF-Tu will bind to the ribosome.     

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.     

Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4

Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 μM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 μM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.