Expression of recombinant elongation factor 1 beta from rabbit in Escherichia coli. Phosphorylation by casein kinase II

Chen, Charng‐Jui; Traugh, Jolinda A.

doi:10.1016/0167-4781(95)00166-2

Cited by 11 publications

(9 citation statements)

References 26 publications

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“…Clearly, EF-1␤ plays an important role in the efficiency and accuracy of the translation elongation process. Further genetic and biochemical analysis of these mutants will provide insight into factors that may regulate translation elongation, such as kinases (7,16,29), or interactions with other components of the translational apparatus such as the ribosome or release factors.…”

Section: Fig 6 Suppression Of the Csmentioning

confidence: 99%

Mutations in Elongation Factor 1β, a Guanine Nucleotide Exchange Factor, Enhance Translational Fidelity

Carr-Schmid

Valente

Loik

et al. 1999

Molecular and Cellular Biology

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Translation elongation factor 1␤ (EF-1␤) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1␤ is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1␣, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1␤ is sufficient for normal cell growth. This region of yeast and metazoan EF-1␤ and the metazoan EF-1␤-like protein EF-1␦ is highly conserved. Human EF-1␤, but not human EF-1␦, is functional in place of yeast EF-1␤, even though both EF-1␤ and EF-1␦ have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1␤ protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1␤ in regulating EF-1␣ activity, cell growth, translation rates, and translational fidelity.Translation elongation requires the function of soluble protein factors. In eukaryotic organisms, the translation elongation factor 1 (EF-1) delivers aminoacyl-tRNAs (aa-tRNAs) to the A site of an elongating ribosome (6). EF-2 functions after peptide bond formation to translocate the peptidyl-tRNA to the P site (30). EF-3 is a third fungus-specific elongation factor (3). All three factors have a requirement for energy from either GTP or ATP. Only EF-1, however, requires a nucleotide exchange factor to maintain the pool of active nucleotide triphosphate-bound protein.EF-1 is composed of four subunits in metazoans (␣, ␤, ␥, and ␦) (6) but only three subunits in the yeast Saccharomyces cerevisiae (␣, ␤, and ␥) (31). The ␣ subunit is a classic G protein that binds aa-tRNAs in a GTP-dependent manner and delivers them to the A site of the elongating ribosome. After GTP hydrolysis, the resulting GDP remains bound to EF-1␣, and the protein is unable to reenter the elongation cycle until GTP is rebound. The EF-1␤ subunit functions as a guanine nucleotide exchange factor in vitro (38). While EF-1␤ is found associated with the ␥ subunit, the role of EF-1␥ remains unknown, although it modestly stimulates the activity of EF-1␤ in vitro (41). The ␦ subunit of metazoans also functions as a guanine nucleotide exchange factor in vitro but may function in vivo in the assembly of higher-order complexes of EF-1 with aa-tRNA synthetases (2). There is no biochemical or genetic evidence that yeasts contain an EF-1␦ subunit.The ␤ subunit of yeast EF-1 (yEF-1) is encoded by the single-copy essential gene TEF5 (14). The requirement for yEF-1␤ can be relieved by the presence of an extra copy of the gene encoding yEF-1␣ (20). The resulting yEF-1␤-deficient stra...

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Section: Fig 6 Suppression Of the Csmentioning

confidence: 99%

Mutations in Elongation Factor 1β, a Guanine Nucleotide Exchange Factor, Enhance Translational Fidelity

Carr-Schmid

Valente

Loik

et al. 1999

Molecular and Cellular Biology

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“…In other studies, EF-1␤ from Artemia, rabbit and wheat germ was shown to be phosphorylated by CKII (24). Using recombinant EF-1␤ from rabbit, Ser 106 and Ser 112 in the sequence of DLFGS 106 DDEEES 112 EEA were phosphorylated by CKII (5).…”

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confidence: 99%

“…Construction of Expression Vectors for EF-1␤, ␥, and ␦-Rabbit EF-1␤ was cloned from a rabbit spleen cDNA library, sequenced, and expressed in E. coli as described by Chen and Traugh (5). The cDNA for EF-1␥, cloned and sequenced from the rabbit spleen cDNA library (13), and the cDNA for rabbit EF-1␦ were subcloned into the pT7-7 expression vector.…”

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confidence: 99%

Recombinant Subunits of Mammalian Elongation Factor 1 Expressed in Escherichia coli

Sheu¹,

Traugh

1997

Journal of Biological Chemistry

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The first step in elongation requires two different activities; elongation factor (EF)-1␣ transfers aminoacyltRNA to the ribosome and is released upon hydrolysis of GTP, EF-1␤␥␦ catalyzes exchange of GDP on EF-1␣ with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the ␤, ␥, and ␦ subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant ␤ subunit migrates as a dimer and the ␥ subunit as a trimer upon gel filtration, whereas the ␦ subunit forms a large aggregate. Complexes of ␤␥, ␥␦ and ␤␥␦ were formed by selfassociation and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between ␤ and ␦. The activity of the recombinant subunits was determined with native EF-1␣ by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant ␤ and ␦ alone stimulated the rate of elongation by 10-fold, with a ratio of 5␣:2␤ or ␦. The ␤␥␦ complex stimulated EF-1␣ activity up to 10-fold with a ratio of 20␣ to 1␤␥␦. Phosphorylation of the ␤ and ␦ subunits alone or in ␤␥␦ by protein kinase CKII had no effect on the rate of elongation. Eukaryotic elongation factor (EF)1 1 consists of four subunits, EF-1␣, ␤, ␥, and ␦. EF-1␣ (50 kDa) forms a ternary complex with GTP and aminoacyl-tRNA and transfers the aminoacyl-tRNA to 80 S ribosomes with the hydrolysis of GTP. EF-1␤␥␦ facilitates the exchange of the GDP bound to EF-1␣ for GTP, initiating another round of elongation (1, 2). EF-1␤ and ␦ contain the GTP exchange activity (3); ␥ is tightly associated to ␤ and is removed only under denaturing conditions (4). The cDNAs for ␤ and ␥ have been cloned and sequenced from a number of different organisms and tissues (5-13). Recently, EF-1␦ cDNA has been sequenced from human (14) and Xenopus (15), and partial amino acid sequences have been obtained from Artemia (14). A leucine zipper motif in the amino terminus of ␦ is found in all three species (14). EF-1␤ and ␦ have a highly homologous carboxyl terminus that contains the GDP/GTP exchange activity, whereas the amino-terminal domains differ and appear to be important for regulation of EF-1 activity. The function of the ␥ subunit is unknown, although there is evidence that ␥ can stimulate the nucleotide exchange activity of ␤ (16). EF-1␥ may anchor the complex to the membrane (16) and has been shown to contain a sequence homologous to glutathione S-transferase in the amino-terminal domain, which has been postulated to be involved in regulation of the assembly of multisubunit complexes (17).Eukaryotic EF-1 has been isolated from a variety of organisms and tissues with molecular weights ranging from 50,000 to about 1 ϫ 10 6 . The low molecular weight form is EF-1␣, the intermediate form is EF-1␣␤␥␦, and the high molecular weight form is a complex of five polypeptides, valyl-tRNA synthetase (ValRS) and .Using purified subunits of EF-1 from Artemia, t...

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“…1A, B). The C-terminal regions of EF-1 b and EF-1d from D. melanogaster, 15) A. salina, 2,6,12) X. laevis, 9,10) rabbit, 13,14) and human 8,11) share greater than 70z amino acid identity, whereas the N-terminal regions share only about 30z amino acid identity. In addition, the C-terminal halves of silk gland EF-1d (amino acid residues 131-262) and EF-1 b (amino acid residues 91-222) are 72.7z identical (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4) The cDNAs for EF-1 b, g, and d subunits from a number of diŠerent organisms and tissues have been cloned and sequenced. [5][6][7][8][9][10][11][12][13][14][15][16] We have cloned the cDNAs encoding the B. mori silk gland EF-1a, b, g, and d subunits, which consist of 463, 222, 423, and 262 amino acids, respectively. [17][18][19] The carboxyl (C)-terminal regions of the EF-1 b and EF-1d subunits (amino acid residues 91-222 and 131-262, respectively) are 72.7z identical in amino acid sequence, but the similarity of their amino-terminal regions is minor (¿30z).…”

mentioning

confidence: 99%

Interaction between Elongation Factors 1β and 1γ fromBombyx moriSilk Gland

Kamiie

Yamashita

Taira

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: a (51 kDa), b (26 kDa), g (49 kDa), and d (33 kDa). The EF-1a subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1a-bound GDP is then exchanged for GTP by the EF-1 bgd complex. To facilitate analysis of the roles of the individual EF-1 b, g, and d subunits in GDP W GTP exchange on EF-1a, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1 b, EF-1g, and the carboxyl-terminal half of EF-1d were expressed, puriˆed, and examined for protein:protein interactions by gelˆltration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1 b and g subunits in a 1:1 molar ratio was detected by gelˆltration. A higher molecular weight species containing an excess of EF-1g relative to EF-1 b was also detected. The amino-terminal region of EF-1 b (amino acid residues 1-129) was su‹cient for binding to EF-1g. The carboxyl-terminal half of EF-1d did not appear to form a complex with EF-1g.

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Expression of recombinant elongation factor 1 beta from rabbit in Escherichia coli. Phosphorylation by casein kinase II

Cited by 11 publications

References 26 publications

Mutations in Elongation Factor 1β, a Guanine Nucleotide Exchange Factor, Enhance Translational Fidelity

Mutations in Elongation Factor 1β, a Guanine Nucleotide Exchange Factor, Enhance Translational Fidelity

Recombinant Subunits of Mammalian Elongation Factor 1 Expressed in Escherichia coli

Interaction between Elongation Factors 1β and 1γ fromBombyx moriSilk Gland

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