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.     

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.     

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.     

Molecular dynamics of ribosomal elongation factors G and Tu

Translation on the ribosome is controlled by external factors. During polypeptide lengthening, elongation factors EF-Tu and EF-G consecutively interact with the bacterial ribosome. EF-Tu binds and delivers an aminoacyl-tRNA to the ribosomal A site and EF-G helps translocate the tRNAs between their binding sites after the peptide bond is formed. These processes occur at the expense of GTP. EF-Tu:tRNA and EF-G are of similar shape, share a common binding site, and undergo large conformational changes on interaction with the ribosome. To characterize the internal motion of these two elongation factors, we used 25 ns long all-atom molecular dynamics simulations. We observed enhanced mobility of EF-G domains III, IV, and V and of tRNA in the EF-Tu:tRNA complex. EF-Tu:GDP complex acquired a configuration different from that found in the crystal structure of EF-Tu with a GTP analogue, showing conformational changes in the switch I and II regions. The calculated electrostatic properties of elongation factors showed no global similarity even though matching electrostatic surface patches were found around the domain I that contacts the ribosome, and in the GDP/GTP binding region.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

EF-G-dependent GTPase on the ribosome. conformational change and fusidic acid inhibition

Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements focus on the distance between the G' region of EF-G and the N-terminal region of L11 (L11-NTD), located within the GTPase activation center of the ribosome. We demonstrate that single-turnover ribosome-dependent EF-G GTPase proceeds according to a kinetic scheme in which rapid G' to L11-NTD movement requires prior GTP hydrolysis and, via branching pathways, either precedes P(i) release (major pathway) or occurs simultaneously with it (minor pathway). Such movement retards P(i) release, with the result that P(i) release is essentially rate-determining in single-turnover GTPase. This is the most significant difference between the EF-G.GTPase activities of vacant and translocating ribosomes [Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) Mol. Cell 11, 1517-1523], which are otherwise quite similar. Both the G' to L11-NTD movement and P(i) release are strongly inhibited by thiostrepton but not by FA. Contrary to the standard view that FA permits only a single round of GTP hydrolysis [Bodley, J. W., Zieve, F. J., and Lin, L. (1970) J. Biol. Chem. 245, 5662-5667], we find that FA functions rather as a slow inhibitor of EF-G.GTPase, permitting a number of GTPase turnovers prior to complete inhibition while inducing a closer approach of EF-G to the GAC than is seen during normal turnover.     

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.     

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.     

A central interdomain protein joint in elongation factor G regulates antibiotic sensitivity, GTP hydrolysis, and ribosome translocation

The antibiotic fusidic acid potently inhibits bacterial translation (and cellular growth) by lodging between domains I and III of elongation factor G (EF-G) and preventing release of EF-G from the ribosome. We examined the functions of key amino acid residues near the active site of EF-G that interact with fusidic acid and regulate hydrolysis of GTP. Alanine mutants of these residues spontaneously hydrolyzed GTP in solution, bypassing the normal activating role of the ribosome. A conserved phenylalanine in the switch II element of EF-G was important for suppressing GTP hydrolysis in solution and critical for catalyzing translocation of the ribosome along mRNA. These experimental results reveal the multipurpose roles of an interdomain joint in the heart of an essential translation factor that can both promote and inhibit bacterial translation.     

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.     

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.     

A thermal ratchet model of tRNA–mRNA translocation by the ribosome

The translocation of tRNA coupled with mRNA in the ribosome is one of important steps during protein synthesis. Despite extensive experimental studies, the detailed mechanism of the translocation remains undetermined. Here, based on previous biochemical, cryo-electron microscopic and X-ray crystallographic studies, a thermal ratchet model is presented for this translocation. In the model, during one elongation cycle of the protein synthesis, two large conformational transitions of the ribosome are involved, with one being the relative rotation between the two ribosomal subunits following the peptide transfer, which is facilitated by the EF-G.GTP binding, and the other one being the reverse relative rotation between the two ribosomal subunits upon EF-G.GTP hydrolysis. The former conformational change plays an important role in ensuring the completion of the release of the deacylated tRNA from the ribosome before tRNA–mRNA translocation. The latter reverse conformational change upon GTP hydrolysis is followed by rapid tRNA–mRNA translocation and Pi release, both of which take place independently of each other. This is consistent with the previous biochemical experimental data. Also, the model is consistent with other available experimental results such as the suppression of EF-G-dependent translocation in cross-linked ribosomes and frameshifting under some conditions.     

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.     

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.     

Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation

L11 protein is located at the base of the L7/L12 stalk of the 50 S subunit of the Escherichia coli ribosome. Because of the flexible nature of the region, recent X-ray crystallographic studies of the 50 S subunit failed to locate the N-terminal domain of the protein. We have determined the position of the complete L11 protein by comparing a three-dimensional cryo-EM reconstruction of the 70 S ribosome, isolated from a mutant lacking ribosomal protein L11, with the three-dimensional map of the wild-type ribosome. Fitting of the X-ray coordinates of L11-23 S RNA complex and EF-G into the cryo-EM maps combined with molecular modeling, reveals that, following EF-G-dependent GTP hydrolysis, domain V of EF-G intrudes into the cleft between the 23 S ribosomal RNA and the N-terminal domain of L11 (where the antibiotic thiostrepton binds), causing the N-terminal domain to move and thereby inducing the formation of the arc-like connection with the G' domain of EF-G. The results provide a new insight into the mechanism of EF-G-dependent translocation.     

Interactions of translational factor EF-G with the bacterial ribosome before and after mRNA translocation

A conserved translation factor, known as EF-G in bacteria, promotes the translocation of tRNA and mRNA in the ribosome during protein synthesis. Here, EF-G.ribosome complexes in two intermediate states, before and after mRNA translocation, have been probed with hydroxyl radicals generated from free Fe(II)-EDTA. Before mRNA translocation and GTP hydrolysis, EF-G protected a limited set of nucleotides in both subunits of the ribosome from cleavage by hydroxyl radicals. In this state, an extensive set of nucleotides, in the platform and head domains of the 30S subunit and in the L7/L12 stalk region of the 50S subunit, became more exposed to hydroxyl radical attack, suggestive of conformational changes in these domains. Following mRNA translocation, EF-G protected a larger set of nucleotides (23S rRNA helices H43, H44, H89, and H95; 16S rRNA helices h5 and h15). No nucleotide with enhanced reactivity to hydroxyl radicals was detected in this latter state. Both before and after mRNA translocation, EF-G protected identical nucleotides in h5 and h15 of the 30S subunit. These results suggest that h5 and h15 may remain associated with EF-G during the dynamic course of the translocation mechanism. Nucleotides in H43 and H44 of the 50S subunit were protected only after translocation and GTP hydrolysis, suggesting that these helices interact dynamically with EF-G. The effects in H95 suggest that EF-G interacts weakly with H95 before mRNA translocation and strongly and more extensively with this helix following mRNA translocation.     

Binding of the 3'' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.     

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.     

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.