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William C. Merrick - One of the best experts on this subject based on the ideXlab platform.

  • Purification and Characterisation of a Tissue Specific Elongation Factor 1 Alpha (EF-1α2) from Rabbit Muscle
    Biochemical and biophysical research communications, 1998
    Co-Authors: Peter Kristensen, Ann Lund, Brian F.c. Clark, Jens Cavallius, William C. Merrick
    Abstract:

    Abstract The peptide Elongation Factor 1 alpha (EF-1α) 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-1α 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-1α 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-1α2, is translated. Preliminary activity assays show that the isoform has the same functional activities as the normal EF-1α, designated EF-1α1, in relation to protein synthesis, but may behave differently in the ability to bind nucleotides. Based on the availability of the isoforms of EF-1α purified from a mammalian species, it will be possible to conduct further comparative studies in order to elucidate the different functions of EF-1α1 and EF-1α2 proteins.

  • The Elongation Factor 1 A-2 isoform from rabbit: Cloning of the cDNA and characterization of the protein
    Nucleic acids research, 1998
    Co-Authors: Søren Kahns, Peter Kristensen, Ann Lund, Brian F.c. Clark, Jens Cavallius, Charlotte R. Knudsen, William C. Merrick
    Abstract:

    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.

  • A model for the aminoacyl-tRNA binding site of eukaryotic Elongation Factor 1 alpha.
    The Journal of biological chemistry, 1992
    Co-Authors: Terri Goss Kinzy, John P. Freeman, Arthur E. Johnson, William C. Merrick
    Abstract:

    Abstract Eukaryotic Elongation Factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic Factor EF-Tu, for which an x-ray crystal structure is available.

Wim Möller - One of the best experts on this subject based on the ideXlab platform.

  • Valyl-tRNA synthetase from Artemia Purification and association with Elongation Factor 1
    European journal of biochemistry, 1995
    Co-Authors: Margreet Brandsma, George M. C. Janssen, Pierre Kerjan, Jan Dijk, Wim Möller
    Abstract:

    Two components of the protein biosynthetic machinery, valyl-transfer RNA synthetase (VRS) and Elongation Factor 1 (EF-1), have been isolated as a complex from several mammalian tissues. However, yeast VRS, which lacks an amino-terminal extension, does not associated with EF-1. We purified VRS from the brine shrimp Artemia and investigated its interaction with EF-1. Western blotting of crude Artemia extracts revealed the presence of two forms of VRS, differing in size and capacity to associate with EF-1. About 80% of the total VRS corresponds to a polypeptide of 130 kDa which behaves as a monomer upon gel filtration. Only the larger form of 140 kDa coelutes, cosediments and co-immunoprecipitates with the EF-1 alpha 2 beta gamma delta complex. The ratio of the two forms of VRS remains constant throughout early development. The possible origin and mode of expression of the two forms of VRS present in Artemia are discussed.

  • The subunit structure of Elongation Factor 1 from Artemia. Why two alpha-chains in this complex?
    The Journal of biological chemistry, 1994
    Co-Authors: George M. C. Janssen, H. T. F. Van Damme, Jan Kriek, Reinout Amons, Wim Möller
    Abstract:

    Elongation Factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the Elongation phase of protein biosynthesis. Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex. We describe here the complete subunit structure of Elongation Factor 1, and discuss its change during development of Artemia. Elongation Factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1. Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha 2 beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached. Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the Elongation step of prokaryotic protein synthesis.

  • A major substrate of maturation promoting Factor identified as Elongation Factor 1 beta gamma delta in Xenopus laevis.
    The Journal of biological chemistry, 1991
    Co-Authors: George M. C. Janssen, J. Morales, A. Schipper, J.-c. Labbe, O. Mulner-lorillon, R. Belle, Wim Möller
    Abstract:

    Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic Elongation Factors (EF) 1 beta and 1 gamma from Artemia salina (Belle, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate Elongation Factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on Elongation Factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis.

Marsha L. Frazier - One of the best experts on this subject based on the ideXlab platform.

  • Overexpression of an Elongation Factor‐1 γ‐hybridizing rna in colorectal adenomas
    Molecular carcinogenesis, 1993
    Co-Authors: Bulent Ender, Patrick M. Lynch, Yeul Hong Kim, Nikhil V. Inamdar, Karen R. Cleary, Marsha L. Frazier
    Abstract:

    While it is apparent that colorectal carcinogenesis results from a series of genetic alterations manifested phenotypically by the adenoma-to-carcinoma sequence, the early events that occur in the process of tumorigenesis have not been elucidated. We previously demonstrated that human Elongation Factor-1 (EF-1)γ-hybridizing RNA was overexpressed in 25 of 29 colorectal carcinomas. To determine if the overexpression of this mRNA occurs early in tumor development, we examined 25 adenomas and corresponding normal-appearing distant mucosae from 20 patients without familial adenomatous polyposis (FAP). We observed overexpression at a level of twofold or more in 14 (56%) of the 25 adenomas, indicating that overexpression of EF-1γ RNA is often a relatively early event in the development of non-FAP colorectal cancer.

  • Expression of Elongation Factor-1 gamma-related sequence in human pancreatic cancer.
    Pancreas, 1992
    Co-Authors: Young Lew, Dennie V. Jones, Wendy Mars, Douglas B. Evans, David R. Byrd, Marsha L. Frazier
    Abstract:

    A cDNA clone designated pPDC-1 was isolated from a cDNA library prepared against poly(A+)RNA isolated from the human pancreatic adenocarcinoma cell line, Capan-2. The cDNA corresponds to a 1.7-kilobase mRNA that is expressed at higher levels in seven of nine pancreatic tumors than in their corresponding normal tissues. It is also expressed in normal human kidney, intestine, pancreas, stomach, placenta, lung, brain, spleen, and liver. A computer search of the Intelligenetics System of all available nucleotide sequences revealed a 60% homology between the nucleotide sequence of the pPDC-1 cDNA and that of Elongation Factor-1 gamma from Artemia. The deduced amino acid sequence shared 53% identity with the amino acid sequence for the Artemia Elongation Factor-1 gamma.

  • Expression of an Elongation Factor 1 γ-related sequence in adenocarcinomas of the colon
    Gastroenterology, 1992
    Co-Authors: Kefung Chi, Dennie V. Jones, Marsha L. Frazier
    Abstract:

    Abstract The authors have previously reported that a messenger RNA (mRNA) bearing 60% homology to Elongation Factor 1 γ in Artemia salina was overexpressed in 7 of 9 pancreatic tumors relative to normal appearing adjacent tissue. The purpose of the present study was to determine if this pattern of overexpression is also detected in colorectal carcinoma. Overexpression was observed in 25 of 29 colorectal carcinomas, relative to normal adjacent tissue. Of them, mRNA was overexpressed in 2 tumors classified as Dukes' D, in 8 of 11 tumors classified as Dukes' B2, and in 15 of 16 tumors classified as Dukes' C2.

D. C. H. Yang - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of a novel N-terminal peptide in human aspartyl-tRNA synthetase. Roles in the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the Elongation Factor 1 alpha.
    The Journal of biological chemistry, 1994
    Co-Authors: V. S. Reed, D. C. H. Yang
    Abstract:

    Abstract The kinetics of the N-terminal 32 residue-deleted human aspartyl-tRNA synthetase (hDRS delta 32) was analyzed. The kinetics of aspartyl-adenylate formation and Asp-tRNA synthesis by hDRS delta 32 were indistinguishable from those of hDRS. However, the dissociation of Asp-tRNA from hDRS delta 32 was much faster than that of hDRS. Unlike hDRS delta 32-catalyzed aspartylation of tRNA was not affected by the Elongation Factor 1 alpha. Two N-terminal peptides of hDRS, hDRS(T5-E26) and hDRS(D12-R27), were synthesized. Both peptides bind to tRNA-Sepharose. Both peptides, hDRS(T5-E26) and hDRS(D12-R27), are monomeric and oligomerize at high peptide concentration or in 50% propylene glycol. The peptide hDRS(T5-E26) showed little alpha-helical content as analyzed by CD spectroscopy, while hDRS(D12-R27) showed appreciable alpha-helical contents in nonpolar solvents. These results suggest that the N terminus in hDRS may mediate the slow release of Asp-tRNA and facilitate the interaction of the hDRS.Asp-tRNA complex with the Elongation Factor 1 alpha. The demonstration of alpha-helix formation of the hDRS N-terminal peptide is consistent with the hypothetical amphiphilic helix of the N-terminal extension in hDRS. A model for the transfer of Asp-tRNA from hDRS to Elongation Factor 1 alpha is presented.

  • Mechanisms of the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the Elongation Factor 1 alpha
    The Journal of biological chemistry, 1994
    Co-Authors: V. S. Reed, Meryl E. Wastney, D. C. H. Yang
    Abstract:

    Aspartylation of mammalian tRNAAsp by bacteria-expressed human aspartyl-tRNA synthetase (hDRS) was examined. The kinetics of the aspartylation of tRNA was consistent with the following reaction pathway, [formula: see text] where E, represents aspartyl-tRNA synthetase. A set of rate constants was obtained which fit single turnover time courses at varying concentrations of the enzyme, tRNA, and AMP using the SAAM program. The dissociation of Asp-tRNA (k3) was found to be rate limiting. The Elongation Factor 1 alpha (EF1 alpha) and GTP stimulated the hDRS aspartylation. The stimulation depended on the presence of both EF1 alpha and GTP. Kinetic analysis indicated that EF1 alpha formed a complex with the hDRS-Asp-tRNA complex and stimulated the dissociation of Asp-tRNA. In the presence of 0.5 M NH4Cl, which enhances the binding of Asp-tRNA by EF1 alpha, hDRS-bound Asp-tRNA can be transferred directly to EF1 alpha. The implications of these results on the function of the multi-tRNA synthetase complex will be discussed.

Anna V. El'skaya - One of the best experts on this subject based on the ideXlab platform.

  • EUKARYOTIC TRANSLATION Elongation Factor 1 ALPHA : STRUCTURE, EXPRESSION,FUNCTIONS, AND POSSIBLE ROLE IN AMINOACYL-TRNA CHANNELING
    Progress in nucleic acid research and molecular biology, 1998
    Co-Authors: Boris Negrutskii, Anna V. El'skaya
    Abstract:

    This review offers a comprehensive analysis of eukaryotic translation Elongation Factor 1 (eEF-1 alpha) in comparison with its bacterial counterpart EF-Tu. Altogether, the data presented indicate some variances in the Elongation process in prokaryotes and eukaryotes. The differences may be attributed to translational channeling and compartmentalization of protein synthesis in higher eukaryotic cells. The functional importance of the EF-1 multisubunit complex and expression of its subunits under miscellaneous cellular conditions are reviewed. A number of novel functions of EF-1 alpha, which may contribute to the coordinate regulation of multiple cellular processes including growth, division, and transformation, are characterized.

  • Rabbit translation Elongation Factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase.
    FEBS letters, 1996
    Co-Authors: Boris Negrutskii, T V Budkevich, V F Shalak, G V Turkovskaya, Anna V. El'skaya
    Abstract:

    Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964-968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation Elongation Factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found. The effect is shown to be specific towards the origin of tRNA and Elongation Factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis.