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.     

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.     

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.     

Increase of fidelity of polypeptide synthesis by spermidine in eukaryotic cell-free systems

The mechanism of spermidine-induced increase of fidelity of polypeptide synthesis in a wheat germ cell-free system has been studied. It was found that the increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl-tRNA to ribosomes, that reduction of misreading was more marked at the 5'-base than at the 3'-base of the codon and that misreading caused by paromomycin and kanamycin C was not significantly decreased by spermidine. It was deduced from these results that spermidine inhibited low-frequency misreading more strongly than high-frequency misreading. In addition, spermidine was found to stimulate the rejection of non-cognate aminoacyl-tRNA mainly at an initial discrimination step during the binding of amino-acyl-tRNA to ribosomes, and slightly at a subsequent GTP-dependent discrimination step, the so-called proofreading step. In yeast, rabbit reticulocyte, and Artemia salina cell-free systems, spermidine was found to increase the fidelity of protein synthesis.     

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.     

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.     

Inhibition by Thiopeptin of Bacterial Protein Synthesis

Thiopeptin, a sulfur-containing antibiotic, was found to inhibit protein synthesis in a bacterial ribosomal system. The pretreatment of ribosomal subunits with the antibiotic revealed that thiopeptin may act on the 50 S ribosomal subunit. The elongation of peptide chain on the ribosome is more profoundly blocked by the antibiotic than the initiation of protein synthesis. It was demonstrated that thiopeptin inhibits elongation factor (EF)-Tu-dependent GTP hydrolysis and binding of aminoacyl-tRNA to the ribosome. The peptidyl transferase-catalyzed puromycin reaction is not significantly affected by the antibiotic. Thiopeptin inhibits EF-G-associated GTPase reaction, and translocation of peptidyl-tRNA and mRNA from the acceptor site to the donor site. Protein synthesis in ribosomal systems, obtained from rat liver and rabbit reticulocytes are insensitive to the antibiotic.     

GTPases of the Translation Apparatus

Protein biosynthesis is a complex biochemical process involving a number of stages at which different translation factors specifically interact with ribosome. Some of these factors belong to GTP-binding proteins, or G-proteins. Due to their functioning, GTP is hydrolyzed to yield GDP and the inorganic phosphate ion P_i. Interaction with ribosome enhances GTPase activity of translation factors; i.e., ribosome plays a role of GTPase-activating protein (GAP). GTPases involved in translation interact with ribosome at every stage of protein biosynthesis. Initiation factor 2 (IF2) catalyzes initiator tRNA binding to the ribosome P site and subsequent binding of the 50S subunit to the initiation complex of the 30S subunit. Elongation factor Tu (EF-Tu) controls aminoacyl-tRNA delivery to the ribosome A site, while elongation factor G (EF-G) catalyzes translocation of the mRNA-tRNA complex by one codon on the ribosome. Release factor 3 (RF3) catalyzes the release of termination factors 1 or 2 (RF1 or RF2) from the ribosomal complex after completion of protein synthesis and peptidyl-tRNA hydrolysis. The functional properties of translational GTPases as related to other G-proteins, the putative mechanism of GTP hydrolysis, structural features, and the functional cycles of translational GTPases are considered.     

The inhibition of elongation factor 1 activity by heparin

The effect of the mucopolysaccharide heparin on elongation factor 1 (EF-1) from embryos of the brine shrimp Artemia salina was investigated. Heparin was found to be a potent inhibitor of the purified enzyme in binding aminoacyl-tRNA to ribosomes and had a comparable effect on polyuridylic acid dependent polyphenylanine synthesis. However, no effect on the binding of GTP to EF-1 or the ability of the factor to form a ternary complex with GTP and aminoacyl-tRNA was observed, suggesting that heparin interferes with the ribosome-attachment site on the ternary complex. In addition EF-1 bound to heparin-Sepharose gels and such gels could be used to partially purify the factor from post-ribosomal supernatant fractions.     

Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis.

Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers. Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli. Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal. Moreover, with phage RNA these concentrations increased the synthesis of large polypeptides. We conclude that binding of gentamicin to its first site causes inhibition but little misreading; binding to additional site(s) partly reverses the inhibition by first-site binding and markedly stimulates misreading, and the misreading appears to favor "readthrough" of termination codons. In the third phase (greater than 100 micron) synthesis is slowed again but the pattern of misreading does not appear to be altered; this effect need not involve a specific further action on the ribosome.     

Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation

Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye. EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC). Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation. This finding suggests that their histidines, i.e. His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor.     

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.     

Structural homology between elongation factors EF-Tu from Bacillus stearothermophilus and Escherichia coli in the binding site for aminoacyl-tRNA

Elongation factor EF-Tu (Mr approximately equal to 50 000) and elongation factor EF-G (Mr approximately equal to 78 000) were isolated from Bacillus stearothermophilus in a homogeneous form. The ability of EF-Tu to participate in protein synthesis is rapidly inactivated by N-tosyl-L-phenyl-alanylchloromethane (Tos-PheCH2Cl). EF-Tu X GTP is more susceptible to the inhibition by Tos-PheCH2Cl than is EF-Tu X GDP. Tos-PheCH2Cl forms a covalent equimolar complex with the factor by reacting with a cysteine residue in its molecule. The labelling of EF-Tu by the reagent irreversibly destroys its ability to bind aminoacyl-tRNA, which in turn protects the protein from this inactivation. This indicates that the modification of EF-Tu by Tos-PheCH2Cl occurs at the aminoacyl-tRNA binding site of the protein. To identify and characterize the site of aminoacyl-tRNA binding in EF-Tu, the factor was labelled with [14C]Tos-PheCH2Cl, digested with trypsin, the resulting peptides were separated by high-performance liquid chromatography and the sequence of the radioactive peptide was determined. The peptide has identical structure with an Escherichia coli EF-Tu tryptic peptide comprising the residues 75-89 and the Tos-PheCH2Cl-reactive cysteine at position 81 [Jonák, J., Petersen, T. E., Clark, B. F. C. and Rychlík, I. (1982) FEBS Lett. 150, 485-488]. Experiments on photo-oxidation of EF-Tu by visible light in the presence of rose bengal dye showed that there are apparently two histidine residues in elongation factor Tu from B. stearothermophilus which are essential for the interaction with aminoacyl-tRNA. This is clearly reminiscent of a similar situation in E. coli EF-Tu [Jonák, J., Petersen, T. E., Meloun, B. and Rychlík, I. (1984) Eur. J. Biochem. 144, 295-303]. Our results provide further evidence for the conserved nature of the site of aminoacyl-tRNA binding in elongation factor EF-Tu and show that Tos-PheCH2Cl reagent might be a favourable tool for the identification of the site in the structure of prokaryotic EF-Tus.     

Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation.

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2--GDP--ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.     

How many EF-Tu molecules participate in aminoacyl-tRNA binding and peptide bond formation in Escherichia coli translation?

We have observed that two EF-Tu.GTP cycles are required to make one peptide bond during steady-state translation in an accurate and fast poly(U) translation system prepared from Escherichia coli. We have also found that there are two complexes of EF-Tu.GTP bound to one molecule of aminoacyl-tRNA under our experimental conditions. We suggest, on the basis of these data, that aminoacyl-tRNA enters the ribosomal A-site in a pentameric complex together with two EF-Tu and two GTP molecules. When the tRNA is delivered to the ribosome two GTP molecules are hydrolyzed. It is possible that the functional role of such an EF-Tu dimer is related to the function of the two L7/L12 dimers in the large ribosomal subunit.     

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt''s), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA^Phe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA^His and Phe-tRNA^Phe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt''s and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt''s.     

Factor-free (“Non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes

Systems of poly(U)-directed polyphenylalanino synthesis by Escherichia coli ribosomes in the absence of elongation factors and GTP (factor-free system) or in the presence of one of the elongation factors and GTP (EF-G² and EF-T_u-deperident systems) are described. It is shown that the use of oligouridylates of different length as templates in the factor-free system results in peptides, the degree of polymerization of which does not exceed the number of template codons, i.e. a conjugated translocation of the peptidyl-tRNA and the template takes place. Thus, the function of translocation as well as the specific binding of aminoacyl-tRNA and transpeptidation proved to be intrinsic to the ribosome itself. The study of kinetics of polyphenylalanine synthesis and dependence of the synthesis rate on the Mg²⁺ concentration in the factor-free, EF-T_u-dependent and EF-G-dependent translation systems has demonstrated that the elongation factors with GTP promote ribosomal mechanisms of aminoacyl-tRNA binding and translocation, respectively. It turned out that the factor-free translation system does not display miscoding. It is the promotion of translocation by EF-G with GTP that has been found to be responsible in full measure for miscoding, while EF-T_U with GTP does not contribute to this.     

Affinity labeling of eukaryotic elongation factors using N epsilon-bromoacetyl-Lys-tRNA.

eEF-T and eEF-Tu from rabbit reticulocyte and from Artemia were affinity labeled using N epsilon-bromoacetyl-Lys-tRNA prepared with either yeast or E. coli tRNA. Only the eEF-Tu polypeptide was crosslinked when eEF-T was incubated with the reactive aminoacyl-tRNA analogue, which indicates that at least part of the aminoacyl-tRNA binding site is the same in both eEF-Tu and the multisubunit eEF-T. Complex formation (eEF-Tu x aa-tRNA x GTP) was required for crosslinking, since no covalent reaction with eEF-Tu occurred in the absence of GTP. The yield of crosslinked product was greatly reduced by adding either unmodified rabbit liver aminoacyl-tRNA or unmodified E. coli Lys-tRNA to the incubation to compete for the aminoacyl-tRNA binding site on eEF-T or eEF-Tu, indicating that the covalent reaction occurs while the N epsilon-bromoacetyl-Lys-tRNA is bound in this site. The affinity labeling of a prokaryotic and two different eukaryotic elongation factors by the same reagent suggests that there may be conservation of structure in the region of the proteins which binds the aminoacyl end of the aminoacyl-tRNA.     

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.     

Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies

The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.