Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylation of rat liver RNA polymerase I occurred when intact rat liver nuclei were incubated with [gamma32P]ATP and N6,O2' dibutyryl cyclic 3':5'-AMP. In addition, partially purified RNA polymerase I could be phosphorylated in vitro by an endogenous protein kinase. Phosphorylation by either method was followed by extensive purification of the enzyme. This revealed that 32P remained bound to the enzyme throughout purification. Analysis of the homogeneous labeled protein by polyacrylamide gel electrophoresis under nondenaturing conditions followed by autoradiography revealed that only one of the two forms of RNA polymerase I in rat liver nuclei was phosphorylated. RNA polymerase II was not phosphorylated in intact nuclei. Polyacrylamide gel electrophoresis of the phosphorylated RNA polymerase I in the presence of 0.1% sodium dodecyl sulfate followed by autoradiography demonstrated that the 32P was located primarily on enzyme subunits SA1, SA3, and SA5-SA6. High voltage paper electrophoresis of a partial acid hydrolysate of phosphorylated RNA polymerase I revealed that both serine and threonine residues were phosphroylated. N6,O2'-Dibutyryl cyclic 3':5'-AMP stimulated endogenous RNA polymerase I activity and endogenous nuclear protein phosphorylation in intact nuclei. These results suggest that phosphorylation of RNA polymerase I by nuclear protein kinases may play a role in the control of transcription in mammalian cells.
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PMID:Phosphorylation of rat liver ribonucleic acid polymerase I by nuclear protein kinases. 18 96

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.
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PMID:Purification and characterization of Novikoff ascites tumor protein kinase. 19 79

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.
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PMID:Phosphorylation of calf thymus RNA polymerase II by nuclear cyclic 3',5'-AMP-independent protein kinase. 20 18

Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-T antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 CELLS. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S, with the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind II/III-B showed that all fractions active in the cell-free synthesis of both forms of T-antigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has petides in common with large-T but not with small-T. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result.
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PMID:Cell-free synthesis of simian virus 40 T-antigens. 21

A procedure for the simultaneous purification of RNA polymerases I, II, and III from Saccharomyces cerevisiae is described. High yields of each enzyme activity are obtained, allowing the preparation of approximately 10 mg of polymerase I, 25 mg of polymerase II, and 12 mg of polymerase III from 1.2 kg of cells (wet weight). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates RNA polymerase I contains polypeptides with molecular weights 185 000, 137 000, 41 000, 35 000, 28 000, 24 000, 20 000, 16 000, 14 500, and 12 300; RNA polymerase II contains subunits with molecular weights 170 000, 145 000, 41 000, 33 500, 28 000, 24 000, 18 000, 14 500, and 12 500; and RNA polymerase III contains polypeptides with molecular weights 160 000, 128 000, 82 000, 53 000, 41 000, 37 000, 34 000, 28 000, 24 000, 20 000, 14 500, and 10 700.
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PMID:Isolation of ribonucleic acid polymerases I, II, and III from Saccharomyces cerevisiae. 31 51

Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.
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PMID:Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme. 33 47

DNA-dependent RNA polymerase II (EC 2.7.7.6) from pea seedlings (Pisum sativum var. Alaska) has been purified to homogeneity, as judged by native polyacrylamide electrophoresis. The procedure includes polyethyleneimine precipitation and elution, ammonium sulfate precipitation, DEAE-Sephadex chromatography, phosphocellulose chromatography, and heparin-Sepharose chromatography. The enzyme purified almost to homogeneity has a specific activity of 200 nmol/mg per 15 min at 30 degrees C with denatured calf thymus DNA as template. The enzyme activity is 50% inhibited in the presence of 0.05 migrograms/ml of alpha-amanitin. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that pea RNA polymerase II is composed of eight subunits with molecular weights and molar ratios (in parentheses) of 170 000 (0.9), 140 000 (1.0), 43 000 (1.5), 26 000 (2.0), 22 500 (1.2), 21 500 (0.6), 18 500 (1.6) and 17 500 (2.3). The structure is closely similar to that of cauliflower RNA polymerase II.
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PMID:Purification and subunit structure of RNA polymerase II from the pea. 49 20

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.
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PMID:DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes. 50 Jun 45

DNA dependent RNA polymerase activities in isolated bovine thyroid nuclei and nucleoli have been studied. They retain their RNA synthetic activity for an extended period of time. This RNA synthetic activity is sensitive to actinomycin D and requires the presence of all four ribonucleoside triphosphates. The optimal conditions have been determined. Polyacrylamide gel electrophoresis reveals that the RNA synthesized has a size distribution ranging from 34S to 4S. The production of 18S-8S RNA is very sensitive to low concentrations of alpha-amanitin. However, in isolated bovine thyroid nuclei (not in nucleoli) this drug displays an effect on all RNA classes produced. The alpha-amanitin induced drastic decrease of [3H]-UMP incorporation in RNA of all sizes synthesized by isolated bovine thyroid nuclei is discussed.
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PMID:RNA synthesis in isolated bovine thyroid nuclei and nucleoli. alpha-Amanitin effect, a hint to the existence of a specific regulatory system. 51 Nov 17

An oestrogen receptor was isolated, characterized and purified from the nuclear fraction of the hen oviduct. The receptor sediments at 4.6 S on glycerol gradients, has an equilibrium dissociation constant (Kd) of 1.1 X 10(-10)M, an association constant (ka) of 1.4 X 10(-6) M-1.S-1, and a dissociation constant (kd) of 5 x 10(-5) s-1. The receptor chromatographed from DEAE-cellulose as a single peak at 0.15 M-KCl and was not retained by phosphocellulose. Polyacrylamide-gel electrophoresis of the receptor in the presence of sodium dodecyl sulphate demonstrated two subunits with apparent mol.wts. of 74000 and 80000. The overall purification achieved was 90000-fold by using a combination of cell fractionation, (NH4)2SO4 fractionation and affinity chromatography. This represents the first separation, isolation and purification of the highest-affinity binding component (Kd 10(-10)M) of two high-affinity oestrogen-binding proteins present in both chick and hen oviduct cytosol and nuclei. To examine directly the effect of the purified receptor on transcription a reconstituted cell-free system was used, which contained the receptor--oestradiol complex, Escherichia coli RNA polymerase, rifampicin and chromatin prepared from hormone-withdrawn chick tissue. The receptor-hormone complex at a concentration of 0.1 nM stimulated transcription of oviduct chromatin by promoting an increase of 14000 sites for RNA-chain initiation, which is similar to the number of additional sites measured in the oviducts of diethylstilboestrol-stimulated immature chicks [Tsai, Schwartz, Tsai & O'Malley (1975) J. Biol. Chem. 250, 5165-5174]. Oestradiol alone had no effect on transcription. Thus the data demonstrate that the purified nuclear oestradiol-receptor complex can regulate gene transcription in vitro in a manner similar to that observed in target cells in vivo.
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PMID:Isolation and purification of a hen nuclear oestrogen receptor and its effect on transcription of chick chromatin. 53 32


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