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

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

Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system

The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.     

Activity of the 30-S CsCl core in elongation-factor-dependent GTP hydrolysis.

The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent FTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T-dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent (GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G-GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits.     

The excess GTP hydrolyzed during mistranslation is expended at the stage of EF-Tu-promoted binding of non-cognate aminoacyl-tRNA

The system of translation of Sepharose-bound poly(U) in which all ribosomes are active in peptide elongation was used to determine the stoichiometry of GTP hydrolysis at the stage of EF-Tu-promoted aminoacyl-tRNA binding. The ratio of GTP hydrolyzed at this stage per peptide bond was assayed during codon-specific elongation (polyphenylalanine synthesis) and misreading (polyleucine synthesis). It was demonstrated directly that the excess GTP hydrolyzed during misreading [(1984) FEBS Letters 178, 283-287] is expended at the stage of Ef-Tu-promoted binding of non-cognate aminoacyl-tRNA.     

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.     

Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome

GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation. The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome. Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis. Another conserved arginine, R59, which is homologous to the catalytic arginine of G(alpha) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation. These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different.     

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.     

Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.     

EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome.

Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states. GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation. In addition, large conformational changes have been identified in the ribosome. The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding. Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G.     

The effect of mutations in ribosomal proteins S4, S12 and L7/L12 on EF-Tu-dependent expenditure of GTP in the process of codon-specific elongation and misreading of poly(U)

The effect of mutations in ribosomal proteins S4 (rpsD12), S12 (rpsL282) and L7/L12 (rplL265) of Escherichia coli K12 on the EF-Tu-dependent expenditure of GTP during codon-specific elongation (poly(Phe) synthesis on poly(U] and misreading (poly(Leu) synthesis on poly(U], was studied. Under the conditions used the mutations in proteins S4 and L7/L12 did not practically affect the EF-Tu-dependent expenditure of GTR during the poly(Phe) synthesis on poly(U): the GTP/Phe ratio was about 1, as in the case of the wild strain. Under the same conditions, the ribosomes with a mutant S12 protein tended to discard some amount of Phe-tRNA, as a result of which the GTP/Phe ratio increased to about 3. The marked inhibition of misreading by ribosomes with a mutant S12 protein was accompanied by a significant increase of GTP expenditure at the stage of EF-Tu-dependent non-cognate aminoacyl-tRNA binding. In mutant S 12 proteins the GTP/Leu ratio was about 30-40, whereas in the wild type it was about 12. In contrast, stimulation of misreading by ribosomes with mutant S4 and L7/L12 proteins was accompanied by a decrease of the EF-Tu-dependent expenditure of GTP by 2-3 GTP molecules per one Leu residue included into the peptide.     

The complex formation between Escherichia coli aminoacyl-tRNA, elongation factor Tu and GTP. The effect of the side-chain of the amino acid linked to tRNA

The interaction between Escherichia coli aminoacyl-tRNAs and elongation factor Tu (EF-Tu) x GTP was examined. Ternary complex formation with Phe-tRNAPhe and Lys-tRNALys was compared to that with the respective misaminoacylated Tyr-tRNAPhe and Phe-tRNALys. There was no pronounced difference in the efficiency of aminoacyl-tRNA x EF-Tu x GTP complex formation between Phe-tRNAPhe and Tyr-tRNAPhe. However, Phe-tRNALys was bound preferentially to EF-Tu x GTP as compared to Lys-tRNALys. This was shown by the ability of EF-Tu x GTP to prevent the hydrolysis of the aminoacyl ester linkage of the aminoacyl-tRNA species. Furthermore, gel filtration of ternary complexes revealed that the complex formed with the misaminoacylated tRNALys was also more stable than the one formed with the correctly aminoacylated tRNALys. Both misaminoacylated aminoacyl-tRNA species could participate in the ribosomal peptide elongation reaction. Poly(U)-directed synthesis of poly(Tyr) using Tyr-tRNAPhe occurred to a comparable extent as the synthesis of poly(Phe) with Phe-tRNAPhe. In the translation of poly(A) using native Lys-tRNALys, poly(Lys) reached a lower level than poly(Phe) when Phe-tRNALys was used. It was concluded that the side-chain of the amino acid linked to a tRNA affects the efficiency of the aminoacyl-tRNA x EF-Tu x GTP ternary complex formation.     

The effect of an antiviral peptide on the ribosomal reactions of the peptide elongation enzymes, EF-I and EF-II

A basic peptide with antiviral properties isolated from pokeweed is shown to inhibit the synthesis of globin and phenylalanine peptides on ribosomes isolated from rabbit reticulocytes. The inhibition appears to involve a specific effect of the peptide inhibitor on the larger ribosomal subunit that can be produced at a ratio of inhibitor to ribosomes of less than one to one. Ribosomes treated with the inhibitor have a reduced capacity to support enzymatic binding of Phe-tRNA to ribosomes and GTP hydrolysis caused by the elongation enzyme, EF-I. Treated ribosomes exhibit a concomitant capacity for increased GTP hydrolysis by EF-II but do not efficiently support EF-II-dependent binding of [³H]GTP. Such binding appears to involve the formation of an EF-II·GDP·ribosome complex. Thus, the inhibitor has an effect on GTP-dependent reaction carried out by both of the peptide elongation enzymes. The relation between these effects in the reticulocyte system is discussed in relation to the effects of siomycin or thiostrepton in blocking GTP hydrolysis by EF-T and EF-G on prokaryotic ribosomes.     

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.     

The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes

In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium.     

Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation

On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.     

Mechanism of ribosomal translocation. Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis

The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.     

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.     

Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid

Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.     

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.     

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.