Affinity labelling of the eukaryotic elongation factor EF-2 with the guanosine nucleotide analogue 5′-p-fluorosulfonylbenzoylguanosine

During the translocation of the nascent peptide chain from the ribosomal aminoacyl-site to the peptidyl-site, GTP is hydrolyzed by a mechanism dependent on both ribosomes and the elongation factor EF-2. For insight into the mechanism of GTP hydrolysis, we studied the ability of the GTP analogue 5′-p-fluorosulfonylbenzoylguanosine (FSO₂BzGuo) to act as an affinity label of the guanine-specific site. Pre-incubation of EF-2 with FSO₂BzGuo at increasing concentrations progressively inactivated the EF-2 and ribosome-dependent GTPase activity. Up to 0.5 mM FSO₂BzGuo, the inactivation of the GTPase activity was stoichiometrically correlated with the covalent binding of [³H]FSO₂BzGuo. Thus, one molecule of covalently bound FSO₂BzGuo completely inactivated the GTPase activity of EF-2. Ribosomes or 60-S ribosomal subunits pre-incubated with FSO₂BzGuo were not inactivated, consistent with the idea that the GTP hydrolysis involved in the ribosomal translocation takes place on EF-2.     

On the biological role of ribosomal protein BM-L11 of Bacillus megaterium, homologous with Escherichia coli ribosomal protein L11.

Ribosomes from a thiostrepton-resistant mutant of Bacillus megaterium lack a protein, BM-L11, which is homologous with Escherichia coli ribosomal protein L11. Such ribosomes retain partial activity in cell-free synthesis of polyphenylalanine and can be restored to full activity by reconstitution with protein BM-L11. Examination of individual steps involved in polypeptide chain elongation suggested a role for protein BM-L11, and by inference for E. coli protein L11, in promoting the ribosomal GTP hydrolysis dependent upon elongation factor EF G. Evidently, however, protein BM-L11 is not indispensable for ribosomal function.     

Purification of Euglena EF-1L: A cytoplasmic factor that also functions on procaryotic and organellar ribosomes

The low-molecular-weight form of the cytoplasmic protein synthesis elongation factor-1 (EF-1_L) from Euglena gracilis has been purified extensively from whole-cell extracts. A four-step purification procedure has been developed which results in a 45-fold enrichment in EF-1_L with 10% recovery of the total EF-1 activity present in the post-ribosomal supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the EF-1_L is greater than 90% pure. The purified factor is composed of a single subunit of molecular weight 56,000 as determined by gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. Unlike EF-1s purified to date from other organisms, Euglena EF-1_L catalyzes polymerization on Escherichia coli and Euglena chloroplast ribosomes, as well as on wheat germ ribosomes. The activity of this factor on 70 S ribosomes is about 5% that observed on eucaryotic 80 S ribosomes. This level of catalytic activity is sufficient to obscure the activity of chloroplast EF-Tu and mitochondrial EF-Tu in whole-cell extracts of Euglena. The activity of EF-1_L as measured on either wheat germ or E. coli ribosomes is unstable in the absence of glycerol, is inhibited only slightly by 20 mm, N-ethylmaleimide, is not stimulated by E. coli EF-Ts, and is not inhibited by the antibiotic kirromycin. The relative affinity of EF-1_L for guanine nucleotides was also measured and it was observed that its affinity for GTP is approximately six- to eightfold greater than that for GDP.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.     

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.     

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.     

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.     

Interaction of rabbit reticulocyte elongation factor 1 with guanosine-triphosphate and aminoacyl-transfer ribonucleic acid

Elongation Factor 1 (EF-1) from rabbit reticulocytes interacts with GTP to form a complex that is retained on a nitrocellulose filter. EF-1 also interacts with GDP; however, the concentration of GDP required for maximal complex formation is higher than the concentration of GTP required and the extent of binding is lower. Interaction of EF-1 with GTP in the presence of various aminoacyl-tRNAs from rabbit liver or E. coli results in a 50–75% decrease in the amount of GTP complex retained on a filter. No reduction in the amount of GTP complex retained is observed with deacylated tRNA or with N-acetylphenylalanyl-tRNA. EF-1 is inactivated by heating at 37 °C in the presence of GTP. Aminoacyl-tRNA protects EF-1 from the inactivation observed in the presence of GTP. These data indicate that an interaction of reticulocyte EF-1 with GTP and aminoacyl-tRNA occurs; however, attempts to demonstrate the formation of a stable ternary complex by chromatography on Sephadex G-150 were unsuccessful. Also, no difference is observed between the rate of binding of aminoacyl-tRNA to reticulocyte ribosomes obtained with EF-1 and the rate obtained with EF-1 that had been incubated previously with GTP and aminoacyltRNA.     

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)     

Inhibition of peptide chain termination by antibodies specific for ribosomal proteins

The effects of antibodies specific for the Escherichia coli 30 S and 50 S ribosomal proteins have been determined for in vitro peptide chain termination and two partial reactions, the codon-directed binding of E. coli release factor to the ribosome and peptidyl-tRNA hydrolysis with RF². Antibodies to ribosomal proteins L7 and L12 inhibit the initial binding of RF to the ribosome, and as a result, the subsequent peptidyl-tRNA hydrolysis. The kinetics of ribosomal inactivation for in vitro termination by anti-L7/L12 indicate that Fab fragments bind to three ribosome sites, and suggest that each of three copies of L7/L12 is involved in the binding of RF to the ribosome. When 70 S ribosome substrates are pretreated with anti-L11 and anti-L16 RF-dependent peptidyl-tRNA, hydrolysis is partially inhibited but the interaction of RF with the ribosome is not affected. The inactivation of in vitro termination by a mixture of anti-L11 and anti-L16 is not co-operative. Pretreatment of the 30 S ribosomal subunit (but not 70 S ribosomal substrate) with antibodies to the 30 S proteins, S9 and S11, results in strong inhibition of codon-directed hydrolysis of peptidyl-tRNA. While these antibodies inhibit ribosome subunit association, a requirement for peptide chain termination, and thereby may inhibit the in vitro termination reactions indirectly, the codon-directed binding of RF is markedly more affected than peptidyl-tRNA hydrolysis by anti-S9 and anti-S11. Antibody to S2 and anti-S3 exhibit a similar but less marked differential effect on the partial reactions of in vitro termination under the same conditions. When dissociated ribosomes are pretreated with anti-L11, in vitro termination is completely inhibited and both codon-directed binding of RF and peptidyl-tRNA hydrolysis are affected. L11 may, therefore, be at or near the interface between the ribosome subunits and like S9 and S11 not completely accessible to antibody in 70 S ribosomes. Pretreatment of dissociated ribosomes with antibodies to a number of other ribosomal proteins (L2, L4, L6, L14, L15, L17, L18, L20, L23, L26, L27) results in partial inhibition of all termination reactions although these antibodies have no effect on termination when incubated with 70 S ribosome substrates. The antibodies probably affect in vitro termination indirectly as a result of either preventing correct ribosome subunit association, or preventing correct positioning of the fMet-tRNA at the ribosome P site.     

A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes

An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus. Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion. RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA. These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs. In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit. Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity. This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis. The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region. These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

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.     

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.     

Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.     

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.     

Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.     

Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2

Periodate-oxidized guanine nucleotides (GTPox and GDPox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (greater than 1 h) incubations. The prior reduction EF-2.GTPox with NaBH4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg2+ increased the GTPox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTPox). GTPox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTPox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: Km values found for GTPox and GTP were 7.7 and 3.4 microM, respectively. The binding of GTPox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of GuoPP[CH2]Pox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.     

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.     

Cloning and expression of Bombyx mori silk gland elongation factor 1gamma in Escherichia coli

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of alpha-, beta-, gamma-, and delta-subunits. EF-1alpha GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP. EF-1betagammadelta catalyzes the exchange of EF-1alpha-bound GDP for exogenous GTP and stimulates the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosomes. EF-1gamma cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library. The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively. The silk gland EF-1gamma shares 67.3% amino acid identity with Artemia salina EF-lgamma. The N-terminal domain (amino acid residues 1-211) of silk gland EF-lgamma is 29.3% identical to maize glutathione S-transferase. We demonstrated that silk gland EF-lgamma bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1gamma may have the capacity to bind to glutathione.