Properties and regulation of the GTPase activities of elongation factors Tu and G,and of initiation factor 2

Summary During protein synthesis the interaction with ribosomes of elongation factors Tu (EF-Tu), G (EF-G) and initiation factor 2 (IF-2) is associated with the hydrolysis of GTP which is directly related to the functions of the factors. In this article we review systematically the properties of these GTPase activities in the presence and absence of protein synthesis, and by examining the characteristics of the different minimal systems for the expression of these activities we point to the role of the various effectors and to the enzymological aspects of the systems. For EF-Tu, it has been possible to eliminate any requirement for macromolecular effectors, showing that the factor itself is a GTPase. For EF-G, the presence of at least the 50S ribosomal subunit has remained a requirement, whereas IF-2 needs both the 50S and 30S subunits to exibit GTPase activity. Between the GTPase activities of the three factors there are some striking similarities, but important differences prevail as a consequence of the specificity of the different functions. This can also be seen by examining the respective ribosomal regions implicated in these reactions. When coupled with protein synthesis, the three GTPase activities reveal characteristics differing from those observed in partial systems.     

Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation.

Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli. The truncated factors were produced in a soluble form and retained a high thermostability. It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome-dependent GTPase activity. At the same time, in contrast to the wild-type elongation factor G, they were incapable to promote translocation. The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation. These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation.     

Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.     

GTPase activation of elongation factors Tu and G on the ribosome

The GTPase activity of elongation factors Tu and G is stimulated by the ribosome. The factor binding site is located on the 50S ribosomal subunit and comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the sarcin-ricin loop of 23S rRNA. The role of these ribosomal elements in factor binding, GTPase activation, or functions in tRNA binding and translocation, and their relative contributions, is not known. By comparing ribosomes depleted of L7/12 and reconstituted ribosomes, we show that, for both factors, interactions with L7/12 and with other ribosomal residues contribute about equally and additively to GTPase activation, resulting in an overall 10(7)-fold stimulation. Removal of L7/12 has little effect on factor binding to the ribosome. Effects on other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and translocation, are fully explained by the inhibition of GTP hydrolysis. Based on these results, we propose that L7/12 stimulates the GTPase activity of both factors by inducing the catalytically active conformation of the G domain. This effect appears to be augmented by interactions of other structural elements of the large ribosomal subunit with the switch regions of the factors.     

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.     

Characterization of the ribosomal properties required for formation of a GTPase active complex with the eukaryotic elongation factor 2

The binding stability of the different nucleotide-dependent and -independent interactions between elongation factor 2 (EF-2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF-2- and ribosome-dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF-2.GuoPP[CH2]P with high affinity (Kd less than 10(-9) M) and the rest only capable of binding the factor-nucleotide complex with low affinity (Kd = 3.7 x 10(-7) M). The activity of the EF-2- and 80S-ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF-2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high-affinity complex. At increasing EF-2/ribosome ratios, the ribosomes capable of forming the high-affinity complex was saturated with EF-2, thus allowing formation of the low-affinity ribosome.EF-2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low-affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF-2 in a low-affinity (Kd = 1.3 x 10(-6) M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF-2 in a low-affinity complex. The structural alteration was accompanied by a dramatic increase in the EF-2-dependent GTPase activity. Surprisingly, ricin had no effect on the factor-catalyzed GTP hydrolysis in the presence of 60S subunits alone.     

Soluble factor requirements for the Tetrahymena peptide elongation system and the ribosomal ATPase as a counterpart of yeast elongation factor 3 (EF-3)

Peptide elongation factor 3 (EF-3), which is widely present in yeasts and fungi (Eumycota), does not occur in another lower eukaryote, the unicellular protozoan Tetrahymena pyriformis, as was shown by the following findings: (a) there is no activity to satisfy the EF-3 requirement of yeast ribosomes in the post-ribosomal supernatant fraction from Tetrahymena, and (b) the Tetrahymena ribosomes displayed their full capacity for polyphenylalanine synthesis with purified EF-1 alpha and EF-2 alone from either Tetrahymena or yeast, and their activity on the Tetrahymena ribosomes was not further enhanced by the addition of yeast EF-3, in contrast to the case of the yeast ribosomes. However, as a substitute for the ribosome-activated nucleotidase activity of EF-3, Tetrahymena ribosomes were shown to harbor strong, firmly bound ATPase and GTPase activities, which probably involve the same active site. The ribosome-bound ATPase activity was inhibited by a polyclonal antibody raised against yeast EF-3 with the same inactivation profile as that of polyphenylalanine synthesis on Tetrahymena ribosomes, indicating that the ribosomal ATPase plays an essential role in the elongation process on Tetrahymena ribosomes as previously revealed in the yeast system. It was also shown that the ribosomal nucleotidase plays a pivotal role in the elongation cycle in other eukaryotes.     

Regulation of the activity of eukaryotic peptide elongation factor 1 by autocatalytic phosphorylation

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [gamma-32P]GTP and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF-1 alpha subunit of the factor (Mr 53 000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [gamma-32P]GTP with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32PO4, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21-0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome-independent, acid-labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl-tRNA to 80S ribosomes in the presence of poly(U). This is accompanied by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparently activates its binding site for aminoacyl-tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor-catalyzed binding of aminoacyl-tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF-1 X GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl-tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP-dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis.     

Ribosomal protein L1 from Escherichia coli. Its role in the binding of tRNA to the ribosome and in elongation factor g-dependent gtp hydrolysis

Two Escherichia coli mutants lacking ribosomal protein L1, previously shown to display 40 to 60% reduced capacity for in vitro protein synthesis (Subramanian, A. R., and Dabbs, E. R. (1980) Eur. J. Biochem. 112, 425-430), have been used to study partial reactions of protein biosynthesis. Both the binding of N-acetyl-Phe-tRNA to ribosomes and the 6 to 8-fold stimulation of the elongation factor G (EF-G)-dependent GTPase reaction by mRNA plus tRNA, assayed in the presence of wild type 30 S subunits, were low with L1-deficient 50 S subunits. Addition of pure protein L1 to the assay restored both reactions to 100% of the control. By contrast, the basic EF-G GTPase reaction in the absence of mRNA and tRNA was not at all affected (mRNA alone had no effect). None of the following partial reactions were more than moderately modified by the lack of protein L1: binding to ribosomes of EF-G.GDP plus fusidic acid; the translocation reaction catalyzed by EF-G plus GTP; poly(U)-dependent binding to ribosomes of Phe-tRNAPhe (whether dependent on elongation factor Tu plus GTP or not); and the EF-Tu-dependent GTPase activity. It is concluded that protein L1 is involved in the interaction between ribosomes and peptidyl-tRNA (or tRNA) in the peptidyl site and consequently in the ribosomal GTPase activity depending on the simultaneous action of tRNA and EF-G.     

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.     

Characterization of the ATPase and GTPase activities of elongation factor 3 (EF-3) purified from yeasts

Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe. Both of the proteins consist of a single peptide chain. The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes. The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP. Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2. Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2. Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.     

Binding of eucaryotic elongation factor Tu to nucleic acids

The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated. eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides. The factor binds at multiple sites on 28 S rRNA without strong cooperativity. eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C). Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP. The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes. Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes. In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U). The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction. The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis.     

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.     

Reduced turnover of the elongation factor EF-1 X ribosome complex after treatment with the protein synthesis inhibitor II from barley seeds

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [14C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 X GTP X aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.     

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.     

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli has been determined. Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of the elongation factor and ribosomes. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate ws analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus atom. It is therefore likely that the hydrolysis occurs in a single step by direct, in-line transfer of the phosphorus from GDP to a water oxygen, without a phosphoenzyme intermediate.     

Kinetic determination of the effects of ADP-ribosylation on the interaction of eukaryotic elongation factor 2 with ribosomes

The effect of ADP-ribosylation on the function of eukaryotic elongation factor 2 (EF-2) was investigated by kinetic analysis of the EF-2-catalyzed hydrolysis of GTP in the presence of ribosomes and by direct determination of the affinity of the modified factor for the ribosome. Under conditions where the concentration of EF-2 was rate-limiting, the ADP-ribosylation reduced the maximum rate of GTP hydrolysis and the second order rate constant Kcat/Km by approximately 50%. A similar decrease in Kcat and Kcat/Km was observed when the concentration of ribosomes were kept rate-limiting. The affinity of EF-2 for the pretranslocation type of ribosomes was reduced by 2 orders of magnitude after ADP-ribosylation. No effect was observed in the interaction with the post-translocation type of ribosomes, the ribosomal conformation responsible for activation of the EF-2-dependent GTPase. We conclude that the ADP-ribosylation affects both the association of the modified factor with pretranslocation ribosomes and the hydrolytic capacity of the factor.     

Effect of modeccin on the steps of peptide-chain elongation.

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.     

Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes.

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.     

Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4.The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.