Uncoupled GTPase Research Articles

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Some properties and the possible role of intrinsic ATPase of rat liver 80S ribosomes in peptide bond elongation.

The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.

In Vitro Study of Two Dominant Inhibitory GTPase Mutants of Escherichia coli Translation Initiation Factor IF2: DIRECT EVIDENCE THAT GTP HYDROLYSIS IS NECESSARY FOR FACTOR RECYCLING

We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wild-type IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2.

Regulation of the uncoupled GTPase activity of elongation factor G (EF-G) by the conformations of the ribosomal subunits

The elongation factor G (EF-G) GTPase activity is induced by either 70S ribosomes or 50S ribosomal subunits. The GTPase activity induced by 50S ribosomal subunits is predominant at low concentrations of monovalent cations and decreases with increasing concentrations of K + or NH + 4. Double-logarithmic plots of the data reveal straight lines with different slopes for low and high concentrations of monovalent cations, respectively, intersecting at the same concentration of monovalent cations where maximal EF-G GTPase activity is measured in the presence of both ribosomal subunits. Substantially the same curves are obtained when 50S ribosomal subunits are substituted by 50S CsCl-core particles partially reconstituted by addition of purified 50S split proteins L7/L12. Intact 30S ribosomal subunits, but not 30S CsCl-core particles are able to associate with 50S ribosomal subunits and to modulate ribosome-dependent EF-G GTPase activity. Therefore, our data clearly show that the biphasic courses of the NH + 4 and K + curves of EF-G GTPase activity induced by 50S ribosomal subunits are not due to contaminations with 30S ribosomal subunits but result from different conformations of EF-G/50S ribosomal-subunit complexes at low and high concentrations of monovalent cations, respectively. CD spectra of 50S ribosomal subunits measured under different salt conditions have shown that the conformation of the 50S ribosomal subunits is strongly dependent on the concentration of monovalent cations. The conformation of 30S ribosomal subunits is, however, considerably stronger influenced by the Mg 2+ than by the concentration of monovalent cations. The salt effects on the conformation of the 30S ribosomal subunits correspond to the salt effects on the association of ribosomal subunits and the modulation of EF-G GTPase activity by 30S ribosomal subunits. Since, in the presence of both ribosomal subunits, EF-G GTPase activity is maximal at the same concentration of monovalent cations where obviously a spontaneous conformation change of 50S ribosomal subunits takes place, we postulate that EF-G GTPase primarily acts on the ribosomes by changing the conformation of 50S ribosomal subunits. The resulting model is based on the assumption that EF-G GTPase activity is considerably more strongly induced by the ‘substrate conformation’ (‘state I’) than by the ‘product conformation’ of the 50S ribosomal subunits (‘stateII’). A spontaneous transformation of ‘state I’ to ‘state I’ is expected to occur in the absence of mRNA, aminoacyl-tRNA and EF-T especially under salt conditions favouring state I.

Regulation of elongation factor G GTPase activity by the ribosomal state. The effects of initiation factors and differentially bound tRNA, aminoacyl-tRNA, and peptidyl-tRNA.

The elongation factor G (EF-G) is responsible for the translocation of the ribosome along the mRNA chain. Under in vitro conditions, EF-G exhibits a very active uncoupled GTPase activity which is dependent on the presence of ribosomes and is modulated by mRNA-dependent binding of tRNA. In the absence of tRNA, uncoupled EF-G GTPase is inhibited by initiation factors IF1 and IF3, but not by initiation factor IF2. In the presence of N-fMet-tRNAfMet and poly(A,U,G) or in the presence of N-acetyl-Phe-tRNAPhe and poly(U), initiation factor IF2 causes an additional decrease of the uncoupled EF-G GTPase activity. This effect, however, is dependent on the presence of IF1 and IF3 and is obviously due to the mRNA- and initiation factor-dependent binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, respectively, to the ribosomal P-site. Non-enzymatic binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, however, causes a stimulation of uncoupled EF-G GTPase activity. The same effects are observed for Met-tRNA, Phe-tRNAPhe and uncharged tRNA. These findings are discussed in the light of the three-site model of the ribosome and the mechanism of translocation.

Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes.

The effects of an affinity-purified polyclonal antibody to Artemia salina ribosomal protein L5 on protein synthesis in vitro were examined. The antibody interacted with 60 S subunits more strongly than with 80 S ribosomes, and inhibited reassociation of ribosomal subunits to some extent at 5 mM-Mg2+ but not at 10 mM. Polyphenylalanine synthesis in vitro at 10 mM-Mg2+ was significantly inhibited, especially when the antibody was first preincubated with 60 S subunits prior to the assay. The incorporation of amino acid directed by globin mRNA was inhibited only when the preincubation with 60 S subunits was carried out. On the other hand, no effect was detected on elongation factor 2- and 60 S subunit-dependent uncoupled GTPase activity. These results suggest that L5 is probably located at or near the subunit interface and may play an important role in protein synthesis.

Effect of thiostrepton and 3′-terminal fragments of aminoacyl-tRNA on EF-T u and ribosome-dependent GTP hydrolysis

The effect of the antibiotics thiostrepton and micrococcin on EF-T u-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3′-terminus of aminoacyl-tRNA)(System I) or by methanol (‘uncoupled GTPase’, System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-T u·GTP·70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the K GTP m is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases V GTP max, whereas K GTP m remains unaffected. Micrococcin is without any effect in both systems. The ‘uncoupled GTPase’ (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-T u site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-T u for GTP.

Selective photooxidation of histidine residues in polypeptide chain elongation factor Tu from E. coli.

When EF-Tu was photooxidized for 20 min at 0 degrees C in the presence of 10 microM GDP and 5 microM rose bengal, the activity to promote the binding of [14C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [3H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [14C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA . EF-Tu . GTP complex. Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu . GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J. Biochem. 79, 69-83), was analyzed for histidine content. The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively. Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.

Cation-induced regulatory mechanism of GTPase activity dependent on polypeptide initiation factor 2

Initiation factor IF-2 ribosome dependent GTP hydrolysis (uncoupled GTPase) presents a bell-shaped pH profile which is shifted by changes in ionic strength. At low ionic strength (I = 25 mM) the maximal hydrolytic activity occurs at pH 7.5; when the ionic strength is increased the pH optimum of the reaction is shifted toward more acidic values. Such behavior can be satisfactorily explained as the effect of an electrostatic potential developed by a neighboring polyanion, presumably RNA, on the catalytic site. The addition of fMet-tRNAfMet or AcPhe-tRNAPhe and messenger RNA (coupled GTPase) changes the ionic strength--pH characteristics of the reaction. Thus there is an effect, direct or indirect, of components located at the ribosomal P site. Investigation of the effect of neighboring polyanions on the catalytic activity of the factor-dependent ribosomal GTPases can be seen to provide information about their functional significance that is complementary to that gained from direct structural studies.

Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli. 1. The formation of a ternary guanine-nucleotide . EF-Tu . EF-Ts complex was demonstrated by gel filtration of the T. thermophilus EF-Tu . EF-Ts complex on a Sephadex G-150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli. 2. The dissociation constants for the complexes of T. thermophilus EF-Tu . EF-Ts with GDP and GTP were 6.1 x 10(-7) M and 1.9 x 10(-6) M respectively. On the other hand, T. thermophilus EF-Tu interacted with GDP and GTP with dissociation constants of 1.1 x 10(-9) M and 5.8 x 10(-8) M respectively. This suggests that the association of EF-Ts with EF-Tu lowered the affinity of EF-Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction. 3. Although the T. thermophilus EF-Tu . EF-Ts complex hardly dissociates into EF-Tu and EF-Ts, a rapid exchange was observed between free EF-Ts and the EF-Tu . EF-Ts complex using 3H-labelled EF-Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides. 4. Based on the above findings, an improved reaction mechanism for the regeneration of EF-Tu . GTP from EF-Tu . GDP is proposed. 5. Studies on the functional interchangeability of EF-Tu and EF-Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination. 6. T. thermophilus EF-Ts could bind E. coli EF-Tu to form an EF-Tu (E. coli) . EF-Ts (T. thermophilus hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF-Ts. On the other hand, no stable complex between E. coli EF-Ts and T. thermophilus EF-Tu has been isolated. 7. The uncoupled GTPase activity of T. thermophilus EF-G was much lower than that of E. coli EF-G. T. thermophilus EF-G formed a relatively stable binary EF-G . GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF-G . GDP and EF-G . GTP were 6.7 x 10(-7) M and 1.2 x 10(-5) M respectively. The ternary T. thermophilus EF-G . GDP . ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF-G.

The primary structure of elongation factor G from Escherichia coli Amino acid sequence of the region containing the GTP-binding center

The protein biosynthesis elongation factor EF-G has an exposed SH-group, the modification of which results in the inhibition of the uncoupled GTPase reaction [ 1,2] . This SH-group seems to be contained in the GTP-binding region of EF-G since it was shown in our laboratory that the region including the exposed SH-group is labelled by the photoactivated GTP analogs with a high specificity [3]. Moreover it is known that guanyl nucleotides protect this group from thiol reagents [4]. EF-G incubation with trypsin results in the formation of several fragments comparatively stable to further trypsin treatment [5]. A detailed study of the fragments formed by limited trypsinolysis of EF-G modified by [ 14C ] labeled ICHzCONHz at the exposed SH-group has shown that this SH-group is located in the N-terminal region of EF-G [6]. In this paper we present the amino acid sequence of the fragment containing the exposed SH-group and apparently participating in the formation of the GTPbinding center. The fragment consists of 69 amino acid residues, the only cysteine residue being in position 5.5.

Properties and Function of the Sulfhydryl Group in the Polypeptide Chain Elongation Factor G from E. coli

The properties and function of sulfhydryl groups in the polypeptide chain elongation factor G (EF-G) have been studied by titration with p-chloromercuri[14C]benzoate or 5,5′-dithiobis(2-nitrobenzoic acid), and by assessing the inhibitory effect of N-ethylmaleimide on its activity. EF-G contained three sulfhydryl groups per mole of protein, of which only one was reactive under native conditions. The other two were nonreactive and could be titrated only after complete denaturation of the protein. The activity to catalyze the uncoupled GTPase reaction as well as the ability to form a ternary complex involving EF-G, guanine nucleotides, and ribosomes was completely inactivated by treatment of EF-G with N-ethylmaleimide. The reactive sulfhydryl group was not required for interaction with guanine nucleotides, but was essential for binding to ribosomes. The binary complex involving EF-G and GDP could be isolated after treatment of EF-G with sulfhydryl reagents. The sulfhydryl group of EF-G was not protected by guanine nucleotides alone, but a marked protection was afforded by addition of ribosomes and GMP-P(CH2)P. The reactivity of the sulfhydryl group of EF-G was modulated by interaction with guanine nucleotides. The kinetic studies indicated that reactivity toward N-(1-anilinonaphthyl-4)-maleimide was reduced by GDP and, to a lesser extent, by GTP. These results, together with the previous report on the spin-label probes (Arai, N., Arai, K., Maeda, T., Ohnishi, S., and Kaziro, Y. (1976) J. Biochern. 80, 1057–1065) indicate that (1) the reactive sulfhydryl group of EF-G is required for interaction with ribosomes, and (2) its reactivity is modulated by guanine nucleotide-induced conformational transitions occurring near the reactive protein sulfhydryl.

Mechanism of the Ribosome-dependent Uncoupled GTPase Reaction Catalyzed by Polypeptide Chain Elongation Factor G

At low NH4-+ concentrations, 50S ribosomal subunits from E. coli were fully active in the absence of 30S ribosomal subunits, in forming a complex with the polypeptide chain elongation factor G (EF-G) and guanine nucleotide (ternary complex formation), and also in supporting EF-G dependent hydrolysis of GTP (uncoupled GTPase reaction). However, both activities were markedly inhibited on increasing the concentration of the monovalent cation, and at 160 mM NH4-+, the optimal concentration for polypeptide synthesis in a cell-free system, almost no activity was observed with 50S ribosomes alone. It was found that the inhibitory effect of NH4-+ was reversed by addition of 30S subunits. Thus, at 160 mM NH4-+, only 70S ribosomes were active in supporting the above two EF-G dependent reactions, whereas at 20 mM NH4-+, 50S ribosomes were almost as active as 70S ribosomes. Kinetic studies on inhibition by NH4-+ of the formation of 50S ribosome-EF-G-guanine nucleotide complex, indicated that the inhibition was due to reduction in the number of active 50S ribosomes which were capable of interacting with EF-G and GTP at higher concentrations of NH4-+. The inhibitory effects of NH4-+ on ternary complex formation and the uncoupled GTPase reaction were markedly influenced by temperature, and were much greater at 0 degrees than at 30 degrees. A conformational change of 50S subunits through association with 30S subunits is suggested.

Purification and Characterization of an Inhibitor of Elongation Factor G-dependent Guanosine Triphosphatase Reaction of Ribosomes from Ribosome Wash of Escherichia coli Q13

Abstract An inhibitor of the elongation factor G-dependent uncoupled GTPase reaction of ribosomes has been purified from a solution obtained by washing ribosomes of Escherichia coli Q13 with a buffer containing 0.5 m NH4Cl. The molecular weight of the inhibitor as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration was 23,000 and 19,000, respectively. The inhibitor was specific for the elongation factor G-dependent uncoupled GTPase reaction of 70 S ribosomal particles and did not inhibit the elongation factor G-dependent GTPase reaction of 50 S ribosomal subunit particles. Kinetic studies on the inhibition of the GTPase reaction indicated that the inhibitor was competitive with ribosomes and noncompetitive with elongation factor G and GTP in the GTPase reaction. The ribosomes, which were incubated with the inhibitor and then separated from the free inhibitor, had lower GTPase activity than the ribosomes incubated without the inhibitor. These results are taken as evidence that the inhibitor acts on ribosomes and inhibits the elongation factor G-dependent uncoupled GTPase reaction of ribosomes.

Inhibition of polypeptide chain initiation in Escherichia coli by elongation factor G.

We have previously reported the isolation from E. coli of a specific inhibitor of polypeptide chain initiation that is rendered ineffective when active aminoacylation of transfer RNA is taking place; this is normally the case during natural messenger RNA translation. Surprisingly, the inhibitory activity appears to be a hitherto unrecognized property of the chain elongation factor G. The following hold for preparations purified for either translocase or inhibitor activity: (1) equal electrophoretic mobility on polyacrylamide gels; (2) equal specific activities for (a) inhibition of initiation, (b) translocation, and (c) ribosome-dependent, uncoupled GTPase; and (3) similar heat sensitivity of translocase and inhibitor activities in a temperature-sensitive E. coli mutant with an altered elongation factor G. Different sites are apparently involved in translocation and inhibition because the former, but not the latter, is sensitive to p-chloromercuribenzoate and fusidic acid.

The role of aminoacyl-tRNA binding site on the factor EF-T in uncoupled GTPase reaction

The properties of the N- tosyl- l-phenylalanyl chloromethane treated EF-T factor were studied in a ribosomal system in which splitting of GTP occurs. The action of N- tosyl- l-phenylalanyl chloromethane inhibits the binding of aminoacyl-tRNA to the EF-T factor. The binding and exchange of guanosine phosphates continued to be preserved. The inhibited factor is inactive in the GTPase reaction which depends on the participation of ribosomes and aminoacyl-tRNA. The uncoupled GTPase reaction (which is not dependent on the presence of aminoacyl-tRNA) is also sensitive to the effect of the inhibitor. The inhibition of the uncoupled GTPase is incomplete. These findings are attributed to the involvement of the aminoacyl-tRNA binding site of the EF-T factor in the uncoupled GTPase reaction.

23] Elongation factor T-dependent GTP hydrolysis: Dissociation from aminoacyl-tRNA binding

The elongation factor EFT (Tu + Ts) is required in the mechanism of polypeptide chain elongation for the formation of a ternary complex, aminoacyl-tRNA·EF Tu·GTP, which subsequently binds to the ribosome. In the binding process the GTP molecule is hydrolyzed and a binary complex, EF Tu·GDP, is released. EF Ts displaces the GDP molecule from this complex regenerating the original EFT (Tu + Ts) that can initiate a new cycle. In the presence of methanol, it has been observed that the EF T-dependent GTP hydrolysis, strongly stimulated by the alcohol, is uncoupled from the aminoacyl-tRNA binding to the ribosomes, which is not affected. This ribosome- and EF T-dependent uncoupled GTPase has some interesting characteristics and properties that are described in this chapter.

Kinetic studies on the G-factor-dependent uncoupled GTPase of ribosomes.

Kinetic studies on the G-factor-dependent uncoupled GTPase of ribosomes.

Studies on polypeptide elongation factor from E. coli. I. Crystalline factor G.

The ribosome-dependent guanosine triphosphatase (Elongation Factor G) has been purified from Escherichia coli by protamine precipitation, ammonium sulfate fraction-ation, DEAE-Sephadex column chromatography, gel filtration through Sephadex G-200, and second DEAE-Sephadex column chromatography. The purified factor G was crystallized in the form of needles by addition of ammonium sulfate. The crystalline factor G appears to be homogeneous as judged from ultracentrifugation and gel elec-trophoresis. The sedimentation coefficient of factor G is 4.5 S (so20, W) and the molecular weight as determined from the equilibrium centrifugation is 83,000. Factor G catalyzes the ribosome-dependent hydrolysis of GTP in the absence of messenger RNA and amino-acyl-tRNA (the uncoupled GTPase reaction). Some properties of the uncoupled GTPase reaction are described.

Requirement of an Escherichia coli 50 S Ribosomal Protein Component for Effective Interaction of the Ribosome with T and G Factors and with Guanosine Triphosphate

Abstract Escherichia coli ribosomes were made deficient in a 50 S ribosomal protein component by treatment with ethanol and NH4Cl and their ability to carry out a number of partial reactions involved in polypeptide synthesis was examined. The ribosomes were found to be competent in all reactions in which the ribosome alone participated, but were markedly deficient in all reactions involving an interaction of the ribosome, GTP, and either of the supernatant factors T and G. Nonenzymatic phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA binding and the peptidyl transferase activity were essentially intact in these ribosomes; enzymatic binding of phenylalanyl-tRNA and translocation occur, but at markedly reduced rates; uncoupled GTPase activity associated with G factor and the ability to bind [3H]GTP in the presence of G factor and fusidic acid were virtually abolished; and uncoupled GTPase activity associated with T factor was diminished to about 15% of the activity present with unextracted ribosomes. Polyphenylalanine synthesis and all the partial reactions were completely or substantially restored by readdition of the ribosomal extract.