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A series of mutations in the highly conserved N(153)KMD(156)GTP-binding motif of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro. Two critical regulatory processes of translation elongation, guanine nucleotide exchange and translational fidelity, were analyzed in strains with the N153T, D156N, and N153T/D156E mutations. These strains are omnipotent suppressors of nonsense mutations, indicating reduced A site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in vitro. All three mutant proteins also show an increase in the K(m) for GTP. An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1Balpha (eEF1Balpha) and supported for growth by excess eEF1A was used to show the two mutations with the highest K(m) for GTP restore most but not all growth defects found in these eEF1Balpha deficient-strains to near wild type. An increase in K(m) alone, however, is not sufficient for suppression and may indicate eEF1Balpha performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effectively perform all the functions of eEF1A in vivo.

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Site-directed mutants of eEF1A (formerly eEF-1alpha) were generated using a modification of a highly versatile yeast shuttle vector (Cavallius, J., Popkie, A. P., and Merrick, W. C. (1997) Biochim. Biophys. Acta 1350, 345-358). The nucleotide specificity sequence NKMD (residues number 153-156) was targeted for mutagenesis, and the following mutants were obtained: N153D (DKMD), N153T (TKMD), D156N (NKMN), D156W (NKMW), and the double mutant N153T,D156E (TKNE). All of the yeast strains containing the mutant eEF1As as the sole source of eEF1A were viable except for the N153D mutant. Most of the purified mutant eEF1As had specific activities in the poly(U)-directed synthesis of polyphenylalanine similar to wild type, although with a Km for GTP increased by 1-2 orders of magnitude. The mutants showed a reduced rate of GTP hydrolysis, and most displayed misincorporation rates greater than wild type. The mutant NKMW eEF1A showed unusual properties. The yeast strain was temperature sensitive for growth, although the purified protein was not. Second, this form of eEF1A was 10-fold more accurate in protein synthesis, and its rate of GTP hydrolysis was about 20% of wild type. In total, the wild-type protein contains the most optimal nucleotide specificity sequence, NKMD, and even subtle changes in this sequence have drastic consequences on eEF1A function in vitro or yeast viability.

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Eukaryotic elongation factor 1 A (eEF1A, formerly elongation factor-1 alpha) is an important component of the protein synthesis apparatus. Here we report the isolation and characterization of the cDNA sequence encoding rabbit eEF1A-2, an isoform of eEF1A, as well as a structural and functional comparison of the two rabbit isoforms. Northern analysis of the expression pattern of eEF1A-2 showed that this isoform is expressed in skeletal muscle, heart, brain and aorta, while transcripts are not detected in liver, kidney, spleen and lung. In contrast, the previously characterized eEF1A-1 isoform is expressed in all tissues examined except skeletal muscle. We have recently purified eEF1A-2 from rabbit skeletal muscle. By partial amino acid sequencing and determination of the post-translational modifications of eEF1A-2 we found that both of the glycerylphosphorylethanolamine modifications observed in eEF1A-1 appear to be present in eEF1A-2. However, two of the residues found dimethylated in eEF1A-1 appeared to be trimethylated in eEF1A-2. A comparison of the enzymatic activity showed that eEF1A-1 and eEF1A-2 have indistinguishable activity in an in vitro translation system. In contrast, the GDP dissociation rate constant is approximately 7 times higher for eEF1A-1 than for eEF1A-2. The nucleotide preference ratio (GDP/GTP) for eEF1A-1 was 0.82, while the preference ratio for eEF1A-2 was 1.50.

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The peptide elongation factor 1 alpha (EF-1 alpha) has been isolated and characterised from a number of species. Recently we and others have reported the existence of an isoform of the ubiquitously expressed EF-1 alpha mRNA in higher eukaryotes, including human cells. This isoform has a tissue specific expression pattern, confining it primarily to muscle, heart, and brain. In the present study we have purified the isoform of EF-1 alpha from rabbit muscle. Using partial amino acid analysis, we can conclude that in rabbit muscle essentially only the isoform of elongation factor 1 alpha, designated EF-1 alpha 2, is translated. Preliminary activity assays show that the isoform has the same functional activities as the normal EF-1 alpha, designated EF-1 alpha 1, in relation to protein synthesis, but may behave differently in the ability to bind nucleotides. Based on the availability of the isoforms of EF-1 alpha purified from a mammalian species, it will be possible to conduct further comparative studies in order to elucidate the different functions of EF-1 alpha 1 and EF-1 alpha 2 proteins.

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Eukaryotic elongation factor 1A (eEF1A, formerly eEF-1 alpha) carries aminoacyl-tRNAs into the A-site of the ribosome in a GTP-dependent manner. In order to probe the structure/function relationships of eEF1A, we have generated site-directed mutants using a modification of a highly versatile yeast shuttle vector, which consists of the insertion of a 66 base long synthetic DNA fragment in the vector's polylinker. Via oligonucleotide-directed mutagenesis, the modification permits the identification of mutant clones based on a chromogenic screen of beta-galactosidase activity. Mutagenesis reactions are performed with two or more oligonucleotides, one introducing the chromogenic shift, and the other(s) introducing the mutation(s) of interest in eEF1A. Several rounds of chromogenic shifts and additional mutations can be performed in succession on the same vector. To address the possible function of the methylated lysines in yeast eEF1A, we have changed the post-translationally modified lysines (residue 30, 79, 316 and 390) to arginines using the above methodology. Yeast with eEF1A mutants that substitute arginine in all four sites do not show any phenotypic change. There is also an apparent equivalency of wild-type and mutant yeast eEF1A in in vitro assays. It is concluded that the post-translational modifications of eEF1A are not of major importance for eEF1A's role in translation.

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Elongation factor 1 alpha (EF-1 alpha) is an abundant cellular protein and its amino-acid sequence has been inferred from numerous organisms, including bacteria, archaebacteria, plants and animals. In large measure, it would appear that the overall structure has probably been maintained given the 33% identity and 56% similarity of Escherichia coli EF-Tu with human EF-1 alpha. Chemical sequencing of EF-Tu and EF-1 alpha has revealed that these proteins are post-translationally modified. In order to assess the possible function of these modifications, we have chemically sequenced the EF-1 alpha from the lower eukaryote Saccharomyces cerevisiae (yeast). To our surprise, the methylation pattern of yeast EF-1 alpha was quite different from either rabbit or brine shrimp EF-1 alpha with only the trimethyllysine at position 79 conserved although the yeast protein is 81% identical to rabbit EF-1 alpha. A dimethyllysine was observed at position 316 which corresponds to a trimethyllysine in brine shrimp and rabbit EF-1 alpha. The other positions in yeast EF-1 alpha which were methylated were unrelated to the other six possible positions for modification observed in brine shrimp or rabbit EF-1 alpha. In addition, the unique glyceryl-phosphorylethanolamine observed in mammalian EF-1 alpha and suspected in brine shrimp EF-1 alpha was not found in yeast EF-1 alpha.

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A phosphatidylinositol 4-kinase activator (PIK-A49) has been purified from carrot cells grown in suspension culture. The activator was purified from a soluble fraction using DEAE-Sepharose CL-6B and S-Sepharose chromatography columns. PIK-A49 has a relative molecular mass of 49 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A50 for the activation of the Triton X-100-solubilized phosphatidylinositol 4-kinase fraction was 0.1 microM. Maximal activation was 3-4-fold. The analysis of the sequences of seven peptide fragments containing a total of 142 amino acid residues indicated that PIK-A49 was 69% identical to an actin-binding protein (ABP-50) from Dictyostelium and > 90% identical to elongation factor-1 alpha (EF-1 alpha) from carrot, tomato, and Arabidopsis. PIK-A49 bound actin and facilitated actin polymerization. Poly(U)-directed polyphenylalanine synthesis assays indicated that PIK-A49 had EF-1 alpha activity. The EF-1 alpha activity was enhanced by rabbit EF-1 beta gamma. Activation of phosphatidylinositol 4-kinase by a protein that binds actin and that has EF-1 alpha activity provides additional complexity to the signal transduction mechanisms involving inositol phospholipid metabolism.

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As part of our efforts to characterize eukaryotic translation factors, we have sequenced a number of them chemically and inferred sequences from cDNA clones. To our surprise, there appears to be extensive identity of amino acid sequence in most factors characterized to date in that within mammalian species, usually greater than 99% identity is observed. Extreme examples are rabbit EF-1 alpha which is 100% identical to human EF-1 alpha and rabbit eIF-4AI and eIF-4AII which are 100% identical to mouse eIF-4AI and eIF-4AII for those amino acids sequenced (398/406 and 156/407, respectively). An extended analysis has been made of EF-1 alpha which in rabbit has three different post-translational modifications, dimethyllysine, trimethyllysine and glycerylphosphorylethanolamine. A comparison of the primary structure of EF-1 alpha to E. coli EF-Tu indicates an overall sequence identity of 33%. However, within the amino terminal 180 amino acids (the GTP-binding domain), there are found regions of much greater identity (50/85 = 59%).

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The amount of protein elongation factor EF-2 that can be inactivated by diphtheria toxin-mediated ADP-ribosylation, a measure of its active content, decreases by 45% and 66% in G1-arrested normal human fibroblasts and in HeLa cells respectively. On restimulation of cells with fresh serum, the amounts of ADP-ribosylatable EF-2 begin to increase within 4 h. Whereas the level of active EF-2 returns to normal (exponential phase of growth) in 20 h in the case of fibroblasts, only 47% recovery was observed for HeLa cells during this period. The apparent long half-lives of EF-2 mRNA and protein indicate possibilities of posttranslational ADP-ribosylation and de-ADP-ribosylation as the regulators of the amounts of active EF-2 during human cell cycle.

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A significant decline in amount of active elongation factor, EF-1 alpha, and in its catalytic activity was observed in cell-free extracts prepared from normal human diploid fibroblasts (MRC-5) and their SV40-transformed counterparts, after subjecting the cells to 60 min heat shock at different temperatures. Old MRC-5 cells which had become senescent on serial passaging were more sensitive to heat shock-related changes in activity and amounts of active EF-1 alpha than were rapidly proliferating normal and transformed cells.

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Stoichiometrically estimated amounts of active elongation factor, EF-1 alpha, remain constant in serially passaged Phase II cultures of human fibroblasts, MRC-5, but decrease by 45% towards the end (Phase III) of their lifespan. Catalytic activity of EF-1 alpha is also reduced by 35% in Phase III old cells. The SV40 transformed immortal cell line MRC-5V2 has 30% higher levels of active EF-1 alpha without significant increase in its catalytic activity. Low-serum-associated G1 arrest of normal and transformed cells reduces amounts of active EF-1 alpha by 35% and 20%, respectively. Catalytic activity, however, is reduced rapidly only in G1 arrested normal cells and not in transformed cells. Even though the cell cycle-related changes are reversible both in normal and transformed cells, the age-related decline in amounts of active EF-1 alpha and its activity are irreversible and, most probably, crucial.

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