Role of yeast peptide elongation factor 3 (EF-3) at the AA-tRNA binding step

The stimulatory effect of peptide elongation factor 3 (EF-3), which is uniquely required for the yeast elongation cycle, on the step of binding of aminoacyl-tRNA (AA-tRNA) to ribosomes has been investigated in detail. Yeast EF-1 alpha apparently functions in a stoichiometric manner in the binding reaction of AA-tRNA to the ribosomes. The addition of EF-3 and ATP to this binding system strikingly stimulated the binding reaction, and the stimulated reaction proceeded catalytically with respect to both EF-1 alpha and EF-3, accompanied by ATP hydrolysis, indicating that EF-3 stimulated the AA-tRNA binding reaction by releasing EF-1 alpha from the ribosomal complex, thus recycling it. This binding stimulation by EF-3 was in many respects distinct from that by EF-1 beta gamma. The idea that EF-3 may participate in the regeneration of GTP from ATP and the formed GDP, as indicated by the findings that the addition of EF-3 along with ATP allowed the AA-tRNA binding and Phe polymerization reactions to proceed even in the presence of GDP in place of GTP, was not verified by the results of direct measurement of [32P]GTP formation from [gamma-32P]ATP and GDP under various conditions. Examination of the stability of the bound AA-tRNA disclosed the different binding states of AA-tRNA on ribosomes between in the cases of the complexes formed with EF-1 alpha alone, or factor-independently, and with EF-1 alpha and EF-3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional interaction of yeast elongation factor 3 with yeast ribosomes

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.     

F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction

The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin- binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F- actin, we have studied the effect of F-actin on the ability of EF- 1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa- tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF- 1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus.     

Characterization of the elongation factors from calf brain. 3. Properties of the GTPase activity of EF-1 alpha and mode of action of kirromycin

The GTPase activity of purified EF-1 alpha from calf brain has been studied under various experimental conditions and compared with that of EF-Tu. EF-1 alpha displays a much higher GTPase turnover than EF-Tu in the absence of aminoacyl-tRNA (aa-tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF-1 alpha is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF-Tu, aa-tRNA is much more active than ribosomes in stimulating the EF-1 alpha GTPase activity. However, ribosomes strongly reinforce the aa-tRNA effect. In the absence of aa-tRNA the rate-limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate-limiting, since addition of EF-1 beta enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF-1 alpha; this effect turns into stimulation when aa-tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF-1 alpha/guanine-nucleotide interaction in the presence of ribosomes shows that, differently from EF-Tu, the EF-1 alpha X GDP/GTP exchange reaction takes place on the ribosome.     

Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated nuclear localization

Eukaryotic polypeptide elongation factor EF-1 is not only a major translational factor, but also one of the most important multifunctional (moonlighting) proteins. EF-1 consists of four different subunits collectively termed EF-1alphabeta beta'gamma and EF-1alphabeta gammadelta in plants and animals, respectively. EF-1alpha x GTP catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome. EF-1beta beta'gamma (EF-1beta and EF-1beta'), catalyzes GDP/GTP exchange on EF-1alpha x GDP to regenerate EF-1alpha x GTP. EF-1gamma has recently been shown to have glutathione S-transferase activity. EF-2 catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site on the ribosome. Recently, molecular mimicry among tRNA, elongation factors, releasing factor (RF), and ribosome recycling factor (RRF) has been demonstrated and greatly improved our understanding of the mechanism of translation. Moreover, eukaryotic elongation factors have been shown to be concerned or likely to be concerned in various important cellular processes or serious diseases, including translational control, signal transduction, cytoskeletal organization, apoptosis, adult atopic dermatitis, oncogenic transformation, nutrition, and nuclear processes such as RNA synthesis and mitosis. This article aims to overview the recent advances in protein biosynthesis, concentrating on the moonlighting functions of EF-1.     

Expression of elongation factor 1 beta' in Escherichia coli and its interaction with elongation factor 1 alpha from silk gland.

Silk gland elongation factor 1 (EF-1) consists of four subunits: alpha, beta, beta', and gamma. EF-1 beta beta' gamma catalyzes the exchange of GDP for GTP on EF-1 alpha and stimulates the binding of EF-1 alpha-dependent aminoacyl-tRNA to ribosomes. The carboxy-terminal regions of the EF-1 beta subunits from various species are highly conserved. We examined the region of EF-1 beta' that binds to EF-1 alpha by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1 beta' fusion protein. We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1 beta' for binding to EF-1 alpha.     

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.     

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.     

The mechanism of the protein-synthesis elongation cycle in eukaryotes. Effect of ricin on the ribosomal interaction with elongation factors

The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pre-translocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1-catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 X GTP X aminoacyl-tRNA X ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1- and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.     

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.     

Interaction between elongation factors 1beta and 1gamma from Bombyx mori silk gland

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: alpha (51 kDa), beta (26 kDa), gamma (49 kDa), and delta (33 kDa). The EF-1alpha subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1alpha-bound GDP is then exchanged for GTP by the EF-1betagammadelta complex. To facilitate analysis of the roles of the individual EF-1beta, gamma, and delta subunits in GDP/GTP exchange on EF-1alpha, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1beta, EF-1gamma, and the carboxyl-terminal half of EF-1delta were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1beta and gamma subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1gamma relative to EF-1beta was also detected. The amino-terminal region of EF-1beta (amino acid residues 1-129) was sufficient for binding to EF-1gamma. The carboxyl-terminal half of EF-1delta did not appear to form a complex with EF-1gamma.     

Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.     

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.     

Isolation and characterization of the structural gene encoding elongation factor 3

The unique yeast translational factor EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA to the ribosome. We have isolated the structural gene encoding EF-3 from the yeast Saccharomyces cerevisiae. The YEF3 gene is found in one copy per haploid genome and is essential for vegetative growth. DNA sequence analysis reveals that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence has two repeats of a nucleotide-binding motif. Each of these repeats shows similarity to the nucleotide-binding motif of hydrophilic, membrane-associated ATPases including human multidrug resistant protein MDR. Factor 3 manifests ribosome-dependent ATP hydrolysis. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity and EF-3 protein levels by 3-5-fold. Yeast strains containing elevated EF-3 protein levels also exhibit increased sensitivity to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. These observations suggest that EF-3 may affect translational accuracy.     

New antibiotic that acts specifically on the GTP-bound form of elongation factor Tu.

The new thiazolyl peptide antibiotic GE2270 A, isolated from Planobispora rosea strain ATCC 53773, is shown to inhibit bacterial protein biosynthesis in vitro by affecting specifically the GTP-bound form of elongation factor Tu (EF-Tu). The 'off' rate of EF-Tu.GTP is slowed down 400-fold, locking GTP on EF-Tu, whereas EF-Tu.GDP is unaffected. Therefore, on the EF-Tu.guanine nucleotide interaction, GE2270 A mimicks the effect of aa-tRNA. In line with this, the binding of aa-tRNA to EF-Tu.GTP is hindered by the antibiotic, as shown by the absence of a stable ternary complex and the inhibition of the enzymatic binding of aa-tRNA to the ribosome. This blocks the elongation cycle. GE2270 A does not essentially modify the intrinsic GTPase activity of EF-Tu, but impairs the stimulation by ribosomes of this reaction. The negative effect of GE2270 A on the EF-Tu.GTP interaction with aa-tRNA bears similarities with that of the structurally unrelated pulvomycin, whereas marked differences were found by comparing the effects of these two antibiotics on EF-Tu.GDP. This work emphasizes the varieties of the transitional conformations which tune the EF-Tu interaction with GTP and GDP.     

Effect of cyclic AMP and cyclic GMP on the autophosphorylation of elongation factor 1 from wheat embryos

Extensively purified EF-1H (EF-1 alpha beta beta' gamma) from wheat embryos had a protein kinase activity and phosphorylated EF-1 beta which is one of the two EF-Ts-like factors (EF-1 beta and EF-1 beta'). In this reaction ATP and GTP were equally effective as phosphate donors, and threonine residue was phosphorylated. At 10(-7)M, 3', 5' cyclic GMP stimulated both the phosphorylation and phe-tRNA binding reactions, whereas 3', 5' cyclic AMP inhibited both reactions. These findings indicate that phosphorylation of EF-1H may play an important role in the translational control of protein biosynthesis at the elongation step.     

Binding of reticulocyte elongation factor 1 to ribosomes and nucleic acids.

The present study has examined the requirements for the binding of rabbit reticulocyte elongation factor 1 (EF-1) to ribosomes under different assay conditions. When a centrifugation procedure was used to separate the ribosome EF-1 complex, the binding of EF-1 to ribosomes required GTP and Phe-tRNA, but not poly(U). The results suggested that undr these conditions a ternary complex, EF-1 . GTP . aminoacyl-tRNA, is necessary for the formation of a ribosome . EF-1 complex. However, when gel filtration was used to isolate the ribosome . EF-1 complex, only template and tRNA were required. These studie emphasize the fact that the procedure used to isolate the ribosome . EF-1 complex determines the requirements for stable complex formation. EF-1 can also interact with nucleic acids such as 28 S and 18 S rRNA, messenger RNA and DNA. In contrast to the binding to ribosomes, EF-1 binding to nucleic acids requires only Mg2+.     

Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes.

Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors. In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2). The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA. No significant effect of EF-3 was observed with liver risomes in either assay. In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of [3H]Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin. Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1. No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation. The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization.     

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.     

Interaction of yeast polypeptide chain elongation factor-3 (EF-3) with different nucleotides

ATPase and GTPase activities of EF-3 were similarly inhibited by various nucleotides including CTP, UTP and four dNTP's. The low specificity of EF-3 was in remarkable contrast with the high specificity of EF-1 alpha and EF-2 directed only to quanine nucleotides. The pH-activity and salt concentration-activity profiles as well as the above inhibition experiments coincidently supported that the same active site functions for ATPase and GTPase of EF-3. The stimulation of poly(Phe) synthesis was not observed with AMPPNP in place of ATP. The stimulation required ATP hydrolysis, probably catalyzed by ATPase of EF-3. Reflecting the low specificity of the ATPase, UTP, dTTP, dATP and dGTP stimulated the poly(Phe) synthesis. EF-3 appears to drive yeast elongation cycle using the energy from ATP hydrolysis by its ATPase without serving for GTP regeneration.