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Previously we reported the isolation of a factor, named the R-protein, which strongly repressed RNA polymerase II [EC 2.7.7.6] of Ehrlich ascites tumor cells. In the present work this factor was found to contain much RNA (ratio of RNA to protein, 2.3 to 1). The RNA was G:C rich, with a very high content of guanylic acid (about 38%). On equilibrium density gradient centrifugation in Cs2SO4 solution, the RNA became distributed above free RNA, but after digestion of the R-protein with pronase the RNA cosedimented with free RNA. Thus the R-protein is a complex of RNA and protein. 相似文献
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An improved method was developed for purification of the protein termed S-II that specifically stimulates RNA polymerase II of Ehrlich ascites tumor cells. The specific activity of the final preparation was 400 000 units/mg of protein, which is about 30-fold higher than that of the previous preparation [Sekimizu, K., et al. (1976) Biochemistry 15, 5064]. The final preparation gave a single band on both sodium dodecyl sulfate and nondenaturing gel electrophoresis, and the protein extracted from the band on nondenaturing gel had stimulatory activity. S-II is a basic protein with a molecular weight of 40 500. The fundamental characteristics of S-II determined with the previous preparation were confirmed with completely purified S-II. A specific antibody to S-II was prepared. This antibody inhibited only the stimulatory activity of S-II and did not affect the activity of RNA polymerase II itself. Thus, S-II is probably not a component of the multimeric proteins of RNA polymerase II. 相似文献
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A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations. 相似文献
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Nucleoplasmic RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from calfthymus is phosphorylated by homologous cyclic AMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under non-denaturing conditions revealed that both forms of the enzyme were phosphorylated. Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under denaturing conditions showed that the 25 000 dalton subunit was the phosphate acceptor subunit. Partial acid hydrolysis of the 32P-labeled RNA polymerase II followed by ion-exchange chromatography revealed serine and threonine as the [32P]phosphate acceptor amino acids. Phosphorylation of the RNA polymerase II was accompanied by a stimulation of enzymatic activity and was dependent upon the presence of ATP. 相似文献
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Structural relationships of the three stimulatory factors of RNA polymerase II from Ehrlich ascites tumor cells 总被引:6,自引:0,他引:6
M Horikoshi K Sekimizu S Hirashima Y Mitsuhashi S Natori 《The Journal of biological chemistry》1985,260(9):5739-5744
The structural relationships of S-II, S-II', and S-I(b) stimulatory proteins of RNA polymerase II purified from Ehrlich ascites tumor cells were investigated. From analysis of the amino acid compositions and tryptic peptide maps of these proteins labeled with radioiodinated Bolton-Hunter reagent, it was concluded that S-I(b) is a part of S-II located at either the amino- or carboxyl-terminal and that only this region mainly contains radioiodinatable amino acid residues when labeled using 125I. On chymotryptic digestion, S-II was cleaved to 21- and 18-kDa fragments in the presence of DNA. The 21-kDa fragment was found to be sufficient for stimulation of RNA polymerase II. It was suggested that S-II' is formed by phosphorylation of S-II in the domain containing the 18-kDa fragment. 相似文献
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When partially purified Ehrlich ascites tumor RNA polymerase II was further purified on a column of phosphocellulose, stimulation of its catalysis of RNA synthesis by stimulatory factor S-II was greatly decreased. This decrease in sensitivity to the stimulatory factor was reversible: the enzyme eluted from phosphocellulose became sensitive to the factor when mixed with a protein fraction eluted from the phosphocellulose at high salt concentration. Evidence was obtained that this protein, named helper protein, binds, to the enzyme eluted from phosphocellulose, causing it to recover sensitivity to stimulatory factor S-II. 相似文献
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A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II. 总被引:3,自引:0,他引:3
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T J Guilfoyle 《The Plant cell》1989,1(8):827-836
A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit. 相似文献
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Protein kinase C phosphorylates leukemia RNA polymerase II 总被引:1,自引:0,他引:1
L F Chuang R H Cooper P Yau E M Bradbury R Y Chuang 《Biochemical and biophysical research communications》1987,145(3):1376-1383
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