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.     

Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. III. Temperature-dependent dissociation into subunits in the presence of GTP

Dissociation of highly purified EF-1 alpha beta gamma (a high molecular weight form of polypeptide chain elongation factor-1) from pig liver into EF-1 alpha and EF-1 beta gamma at various temperatures was examined and the following results were obtained. (i) When dissociation of EF-1 alpha beta gamma was analyzed by gel filtration with Sephacryl S-200, it was found that in the absence of GTP, it did not dissociate at any temperature between 4 and 37 degrees C, whereas in the presence of GTP, it tended to dissociate with elevation of the temperature, and almost complete dissociation was observed at 32 degrees C. This indicated that the dissociation constant of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma in the presence of GTP increased with increase in the temperature. (ii) When gel filtration was performed in the presence of both GTP and [14C]Phe-tRNA, the formation of a ternary complex of EF-1 alpha . GTP . [14C]Phe-tRNA from EF-1 alpha beta gamma was noted, and its amount was found to increase with elevation of the temperature. (iii) The amount of [14C]Phe-tRNA bound to ribosomes dependent on added EF-1 alpha beta gamma similarly increased with increase in the temperature, as in the case of ternary complex formation, whereas the binding of [14C]Phe-tRNA to ribosomes dependent on free EF-1 alpha proceeded fairly well even at 0 degrees C. From these results we concluded that among the reaction steps in the binding of [14C]Phe-tRNA to ribosomes dependent on EF-1 alpha beta gamma, dissociation of EF-1 alpha beta gamma to form EF-1 alpha . GTP and EF-1 beta gamma in the presence of GTP is the step which is strongly influenced by temperature.     

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.     

Characterization of elongation factor 1 from yeast

Two species of elongation factor 1 (EF-1) differing in molecular weight have been obtained from the postribosomal supernatant fraction of yeast by chromatography on Sephadex G-200. These two forms are present in approximately equal amounts and both appear to be of cytoplasmic origin. Preparations of the higher and lower molecular weight forms of EF-1 catalyze the poly(U)-directed binding of N-acetylphenylalanylt-RNA (AcPhe-tRNA) to yeast ribosomes. The AcPhe-tRNA binding activity of these preparations is consistently lower than the phenylalanyl-tRNA (Phe-tRNA) binding activity and is more sensitive to N-ethylmaleimide. However, the AcPhe-tRNA binding activity co-purifies with EF-1 on phosphocellulose and has the same heat inactivation profile. Several lines of evidence indicate that the AcPhe-tRNA is bound to the acceptor site of the ribosomes. These and other data strongly suggest that yeast EF-1 is capable of catalyzing the binding of both Phe-tRNA and AcPhe-tRNA to ribosomes.     

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.     

Protein synthesis in yeast. Isolation of variant forms of elongation factor 1 from the yeast Saccharomyces cerevisiae

Two species of the elongation factor 1 (EF-1) differing in molecular weight, subunit composition, and isoelectric point have been isolated from cell-free extracts of the yeast Saccharomyces cerevisiae. The ratio of these two forms of EF-1 activity (EF-1 alpha and EF-1H) seem to vary in different strains and upon the growth phase from which the cells have been isolated. The log phase cells of a protease negative yeast strain EJ101 show a distribution of EF-1 alpha and EF-1H in the ratio of 3:1. Another laboratory yeast strain, D-587-4B, shows a distribution pattern of 4:1. The two forms of EF-1 are completely separable by ion exchange, gel permeation, and hydrophobic and affinity chromatography. Yeast EF-1 alpha is a single polypeptide of molecular weight 50,000 and has an isoelectric point of 8.9. The newly identified form of the yeast EF-1 (EF-1H) has a molecular weight of 200,000. The isoelectric point of this protein is around 5.5. Electrophoresis of the partially purified EF-1H in polyacrylamide gel containing sodium dodecyl sulfate indicates the presence of three nonidentical polypeptides having molecular weights of 50,000, 47,000, and 33,000. The three polypeptides are present in the ratio of 2:1:1. EF-1H is readily converted to EF-1 alpha and EF-1 beta gamma on anion exchange columns. The 50,000 dalton component of EF-1H immunologically cross-reacts with the antibody to EF-1 alpha. The other two polypeptides do not. On the basis of molecular weight, EF-1H is 2-3-fold more active than EF-1 alpha in poly(U)-dependent polyphenylalanine synthesis. EF-1H exchanges nucleotide (GDP----GTP) at a faster rate than EF-1 alpha. Both EF-1 alpha and EF-1H exhibit similar binding constants for GDP and GTP although the affinity of EF-1 alpha for guanine nucleotides is several-fold higher than that of EF-1H. The 33,000-dalton component of EF-1H appears to be functionally analogous to EF-1 beta (Ts) isolated from other eukaryotic sources. The function of EF-1 gamma is unknown.     

Purification and properties of a new polypeptide chain elongation factor, EF-1beta, from pig liver.

Eukaryotic polypeptide elongation factor 1 (EF-1) from pig liver has been resolved into two complementary factors, EF-1alpha and EF-1beta (Iwasaki, K., Mizumoto, K., Tanka, M., and Kaziro, Y. (1973) J. Biochem. (Tokyo) 74, 849). This paper describes the procedures for purification of EF-1beta and some properties of the purified factor. The purification method includes an aqueous two-phase separation technique, a treatment of the crude factor with sodium cholate and two successive column chromatographies on diethyl-aminoethyl-Sephadex A-50. By this method, EF-1beta was purified about 50-fold starting from the material obtained after two-phase separation followed by ammonium sulfate fractionation with a recovery of 20%. The purified EF-1beta appeared homogeneous, having a molecular weight of about 90,000. It consisted of two unequal subunits of the molecular weights of 55,000 and 30,000. It stimulates polymerization of phenylalanine dependent on poly(U) in the presence of both EF-1alpha and EF-2, as well as the EF-1alpha-dependent binding of phenylalanyl-tRNA to ribosomes in the presence of GTP. However, it had no effect on the stoichiometric binding of phenylalanyl-tRNA to ribosomes dependent on EF-1alpha in the presence of guanyl-5'-yl methylenediphosphonate. These results indicate that the function of EF-1beta is to stimulate the recycling of EF-1alpha.     

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+.     

Isolation and properties of elongation factor 1 from Saccharomyces cerevisiae

Polypeptide elongation factor 1 was isolated from yeast postribosomal supernatant. The highly purified factor was resolved on Ultrogel AcA-44 into two complementary fractions. One of these fractions contained two different polypeptide chains corresponding to a Ts-like elongation factor EF-1 beta gamma. The other fraction represented the light form of the factor, designated EF-1 alpha, with a molecular weight of approximately 50,000. The obtained results indicate that EF-1 from lower eukaryotes is also composed of three distinct polypeptides.     

Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits

The guanine nucleotide exchange factor, elongation factor 1 beta gamma (EF-1 beta gamma) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26,000 (EF-1 beta) and 46,000 (EF-1 gamma). A nucleoside diphosphate phosphotransferase activity often found in EF-1 beta gamma preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF-1 beta gamma, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF-1 beta. Both EF-1 beta gamma and EF-1 beta have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF-1 alpha with exogenous GDP; (b) EF-1 alpha-dependent binding of Phe-tRNA to ribosomes; (c) poly(U)-dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF-1 beta gamma compared to EF-1 beta alone. Concerning elongation factor 1 gamma (EF-1 gamma) the following observations were made. In contrast to EF-1 beta, pure EF-1 gamma is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF-1 gamma partitions solely into the detergent phase of Triton X-114 solutions. EF-1 gamma is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF-1 beta gamma was found to be present in membrane fractions, under conditions where no EF-1 alpha was detectable in these fractions. Furthermore it was noted that EF-1 beta gamma copurified strongly with tubulin on DEAE-cellulose. Moreover, it was observed that from a mixture of EF-1 beta gamma and tubulin, EF-1 gamma coprecipitates with tubulin using a non-denaturating immunoprecipitation technique. These findings suggest that EF-1 gamma has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.     

The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.     

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.     

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 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.     

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

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

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.     

Interaction of subunits of polypeptide chain elongation factor I from pig liver. Formation of EF-1alpha.EF-1betagamma and EF-1beta complexes

In the preceding papers, we showed that one of the two complementar factors of polypeptide chain elongation factor 1 (EF-1) from pig liver, EF-1alpha, functionally corresponds to bacterial EF-Tu (Nagata, S., Iwasaki, K., and Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, 168), while the other, EF-1betagamma, as well as one of its subunits, EF-1beta, corresponds to bacterial EF-Ts (Motoyoshi, K. and Iwasaki, K. (1977) J. Biochem. 82, 703). Therefore, the interaction between EF-1alpha and EF-1 betagamma or EF-1beta was was examined and the following results were obtained. i) EF-1betagamma catalytically promoted the exchange of [14C]GDP bound to EF-1alpha with exogenous [3H]GDP. ii). In the absence of the exogenous guanine nucleotide, EF-1betagamma as well as EF-1beta could displace GDP bound to EF-1alpha to form an EF-1alpha.EF-1betagamma as well as an EF-1alpha.EF-1beta complex. iii) The occurrence of EF-1alpha.EF-1betagamma and EF-1alpha.EF-1beta complexes was demonstrated by gel filtration on Sephadex G-150. These results strongly indicate that the mechanism of the action of EF-1betagamma or EF-1beta in converting EF-1alpha.GDP into EF-1alpha.GTP is analogous to bacterial EF-Ts, and the reaction is accomplished by the following reactions; EF-1alpha.GDP + EF-1betagamma (or EF-1beta) in equilibrium EF-1alpha.EF-1betagamma (or EF-1beta) + GDP; EF-1alpha.EF-1beta (or EF-1beta) + GTP IN EQUILIBRIUM EF-1alpha.GTP + EF-1betagamma (or EF-1beta).     

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.     

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.     

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.