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Winston F 《Genome biology》2001,2(2):reviews100-3
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The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha. 相似文献
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Affinity labeling of eukaryotic elongation factors using N epsilon-bromoacetyl-Lys-tRNA. 总被引:3,自引:0,他引:3 下载免费PDF全文
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. 相似文献
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Autoregulation--a common property of eukaryotic transcription factors? 总被引:20,自引:0,他引:20
E Serfling 《Trends in genetics : TIG》1989,5(5):131-133
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Comparison of eukaryotic and prokaryotic elongation factors: isoelectric points and molecular masses
Using the O'Farrell method, a two-dimensional analysis of RNA-binding proteins from rabbit reticulocytes was carried out. The latter have been shown to consist of several scores of polypeptides, predominantly of a moderately basic type with isoelectric points ranging from 7 to 9.5. The two main components of RNA-binding proteins have been identified as eukaryotic elongation factors EF-1L and EF-2. The RNA-binding elongation factors in eukaryotes have higher isoelectric points and somewhat higher molecular masses as compared to their functional analogs from prokaryotes EF-Tu and EF-G having no affinity for RNA. These results are compatible with the assumption that a nonspecific RNA-binding ability of elongation factors in eukaryotes could have arisen in the course of evolution due to the appearance of an additional RNA-binding "domain" of an alkaline type. 相似文献
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Pigott CR Mikolajek H Moore CE Finn SJ Phippen CW Werner JM Proud CG 《The Biochemical journal》2012,442(1):105-118
eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K. 相似文献
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Amoutzias GD Robertson DL Van de Peer Y Oliver SG 《Trends in biochemical sciences》2008,33(5):220-229