Eucaryotic elongation factors Ts is an integral component of rabbit reticulocyte elongation factor 1.

Elongation factor 1 (EF-1) was purified from rabbit reticulocytes and found to contain at least two distinct polypeptides: one of Mr 53 000 and one of Mr 30 000. The 30 000-Mr polypeptide was purified from EF-1 by treatment of the factor with 5.4 M guanidine . HCl and subsequent chromatography on DEAE-BioGel A in the presence of 5 M urea. By a number of functional criteria, the 30 000-Mr polypeptide was found to be the eucaryotic elongation factor Ts (eEF-Ts). These criteria include the ability of the polypeptide to stimulate Artemia salina eEF-Tu-dependent binding of aminoacyl-tRNA to 80-S ribosomes as well as eEF-Tu + EF-2-dependent polyphenylalanine synthesis. The reticulocyte factor also markedly increased the rate of exchange of eEF-Tu . gdp complexes with free GTP. Furthermore, rabbit antibodies to EF-1 from A. salina which was previously shown to contain eEF-Ts [Slobin, L. I. and M?ller, W. (1978) Eur. J. Biochem. 84, 69--77] were found to cross-react with reticulocyte eEF-Ts, suggesting extensive structural homology between brine shrimp and rabbit eEF-Ts. The demonstration that eEF-Ts is and integral component of EF-1 from such diverse sources as brine shrimp and rabbit reticulocytes supports the conclusion that the factor is universally present in eucaryotic EF-1.     

The inhibition of eucaryotic protein synthesis by procaryotic elongation factor Tu

The activity of elongation factor Tu (EF-Tu) from $\text{Escherichia}\text{coli}$ in eucaryotic protein synthesis systems was investigated. EF-Tu was found to inhibit polyphenylalanine synthesis when incubated with $\text{Artemia}$ 80S ribosomes, purified rabbit reticulocyte elongation factor Tu (eEF-Tu) and partially purified reticulocyte translocase enzyme, eEF-G. The inhibition could be overcome by supplying the system with additional eEF-Tu. EF-Tu also inhibited protein synthesis in rabbit reticulocyte lysates. Data presented in this report indicate that inhibition by EF-Tu results from the accumulation of ternary complexes of the protein factor, GTP and aminoacyl-tRNA which do not interact with the ribosomal A-site of 80S ribosomes under physiological conditions.     

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.     

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.     

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.     

The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.

The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.     

Steady state kinetic analysis of the mechanism of guanosine triphosphate hydrolysis catalyzed by Escherichia coli elongation factor G and the ribosome.

The mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor G and the ribosome in the absence of other participants in protein synthesis was examined by steady-state kinetic analysis. Optimal hydrolytic conditions were determined to be approximately pH 8.0, 20 mM Mg2+, and 80 mM NH4+. Kinetic analyses were performed under these conditions at constant elongation factor G concentrations and variable ribosome and GTP concentrations. The resulting double-reciprocal plots in conjunction with the inhibition patterns obtained with GDP indicated that the reaction occurs by an ordered mechanism in which GTP is the leading obligatory substrate. Dissociation constants for GTP and guanosine diphosphate (GDP), as well as limiting Michaelis constants for GTP and ribosomes, were calculated from the double-reciprocal plots. These values are: KSGTP = 37.0 muM, KSGDP = 16.5 muKMGTP = 8.0 muM, KMR = 0.22 muM. Inhibition was also observed at high ribosomal concentrations and suggests that inhibition was due both to the decreased breakdown of the tertiary elongation factor G-GDP-ribosome posthydrolytic complex and to the formation of a nonproductive elongation factor G-ribosome complex. A sequential mechanism with a dead-end elongation factor G-ribosome complex has been constructed to describe the hydrolysis of GTP catalyzed by elongation factor G and the ribosome.     

Association of a GDP binding activity with initiation factor eIF-2 from calf liver

A highly purified preparation of the eucaryotic initiation factor eIF-2 from calf liver which forms a ternary complex with GTP and Met-tRNA_f^Met also exhibits a potent GDP binding activity. The factor preparation specifically forms a binary complex with GDP, other ribonucleoside diphosphates and GTP are inactive. Evidence is presented indicating that the GTP-dependent Met-tRNA_f^Met binding and binary complex formation with GDP are mediated by the same protein which has an apparent molecular weight of 67,000 as judged by glycerol density gradient centrifugation.     

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.     

Kinetic studies on the role of elongation factors 1 beta and 1 gamma in protein synthesis

An equilibrium isotope exchange technique was used to measure in an Artemia system the catalytic influence of elongation factor (EF) 1 beta gamma on the dissociation of GDP from the complex of elongation factor 1 alpha.[3H] GDP in the presence of an excess of free GDP. The kinetic data demonstrate that, in analogy to procaryotes, dissociation of GDP occurs via the formation of a transient ternary complex of EF-1 alpha.GDP.EF-1 beta gamma. The rate constants for the dissociation of GDP from EF-1 alpha.GDP and from the ternary complex EF-1 alpha.GDP.EF-1 beta gamma were found to be 0.7 x 10(-3) and greater than or equal to 0.7 s-1, respectively. The equilibrium association constants of GDP to EF-1 alpha.EF-1 beta gamma and of EF-1 beta gamma to EF-1 alpha.GDP were found to be 2.3 x 10(5) and 4.2 x 10(5) M-1, respectively. Judged from the known elongation rate in vivo and kinetic constants of nucleotide exchange, it was estimated that the recycling of EF-1 alpha may be a rate-controlling step in eucaryotic translation. As a model for GTP exchange, the formation of the ternary EF-1 alpha.guanylyl (beta gamma-methylene)diphosphonate.EF-1 beta gamma complex was also studied. It was observed that both an increase of the level of aminoacyl-tRNA and of temperature favored the dissociation of this complex, thereby enabling EF-1 beta gamma to recycle as a catalyst. This behavior would explain the frequent occurrence of a heavy form of elongation factor 1 in extracts of the eucaryotic cell.     

Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis

A unique feature of eucaryotic adenylate cyclases is their interaction with GTP-binding proteins that mediate hormonal responses. Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein. We describe here that the most abundant protein in E. coli, the GTP-binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity. Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E. coli GTP-binding proteins had no effect on enzyme activity. A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP. To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu-dependent activation of adenylate cyclase is governed by other factors in the cell.     

A conserved amino acid sequence around Arg-68 of Artemia elongation factor 1 alpha is involved in the binding of guanine nucleotides and aminoacyl transfer RNAs

The rate of trypsin cleavage of elongation factor 1 alpha having bound GDP is low and increases on exchange of GDP for GTP. The cleavage occurs at a unique position of the protein chain, namely at arginine-68 of Artemia EF-1 alpha. This increase in trypsin sensitivity is enhanced further in the presence of charged or uncharged transfer RNA. The local unfolding of EF-alpha at residue 68 is discussed in terms of a model in which GTP hydrolysis controls the positioning of a short 3'-terminal section of transfer RNA near the centre of peptide bond synthesis.     

Structure-function relationships in the GTP binding domain of EF-Tu: mutation of Val20, the residue homologous to position 12 in p21.

Val20 of elongation factor Tu (EF-Tu), one of the best-characterized GTP binding proteins, is a variable residue within the consensus motif G-X-X-X-X-G-K involved in the interaction with the phosphates of GDP/GTP. To investigate the structure-function relationships of EF-Tu, which is widely used as a model protein, Val20 has been substituted by Gly using oligonucleotide-directed mutagenesis. The most important effects are: (i) a strong reduction of the intrinsic GTPase activity, (ii) a remarkable enhancement of the association and dissociation rates of EF-TuGly20-GDP, mimicking the effect of elongation factor Ts (EF-Ts) and (iii) the inability of ribosomes to influence the intrinsic GTPase of EF-Tu uncoupled from poly(Phe) synthesis. EF-TuGly20 can sustain poly(Phe) synthesis, albeit at a much lower rate than wild-type EF-TuVal20. As with the latter, poly(Phe) synthesis by EF-TuGly20 is inhibited by the antibiotic kirromycin, but differs remarkably in that it is largely independent of the presence of EF-Ts. According to primary sequence alignment, position 20 is homologous to position 12 of ras protein p21. As in p21, this position in EF-Tu is critical, influencing specifically the GDP/GTP interaction as well as other functions. The effect of the mutation displays diversities but also similarities with the situation reported for p21 having the corresponding residues in position 12. The differences observed with two homologous residues, Gly20 and Gly12 in EF-Tu and p21 respectively, show the importance of a variable residue in a consensus element in defining specific functions of GTP binding proteins.     

Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.     

Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited.     

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.     

An in vitro system derived from Friend erythroleukemia cells to study messenger RNA stability

A system consisting of 40-80S messenger ribonucleoprotein particles (mRNP) from stationary Friend erythroleukemia (FEL) cells was used to investigate the stability of mRNA in vitro. The majority of mRNP mRNAs were found to be stable when incubated for periods of up to ninety minutes at 37 degrees. Nonetheless, many mRNAs are greatly reduced in abundance, including ones for eucaryotic elongation factor Tu (eEF-Tu) and the 73-78 kDa polypeptide commonly found in association with the poly(A) tails of mRNA. A divalent cation dependent ribonuclease (probably an endoribonuclease) could be washed off mRNP by treatment of the particles with 0.5M NaCl. The mRNAs contained in the resultant salt washed mRNPs, including eEF-Tu, were stable when incubated in vitro.     

An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation

The elongation cycle of protein synthesis is completed by translocation, a rearrangement during which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by elongation factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suggests that GTP hydrolysis drives a conformational rearrangement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the energy of GTP hydrolysis is utilized to promote the ribosome rearrangement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA.     

Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (M_r > 500,000) containing four distinct polypeptides (α, β, γ, δ). The other form, EF-1α, consists of a single polypeptide which is the same as the α subunit of EF-1H. Both EF-1α and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1α is enhanced in polyphenylalanine synthesis by a complementary component, EF-1βδ. It is also shown that EF-1βδ can facilitate an exchange of EF-1α-bound GDP for GTP. The EF-1α dissociation constants for GDP and GTP were 0.47 and 0.55 μm respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 μm, respectively. Thus, while EF-1α and EF-1H had approximately the same affinities for GDP and GTP, the EF-1α dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1α or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.