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)

A RNase from calf thymus, which specifically cleaves native or synthetic double-stranded RNA molecules endonucleolytically, has been isolated and purified from calf thymus. For optimal activity, the enzyme requires a sulfhydryl reagent and divalent cations; over 95 per cent of the activity is inhibited by 0.5 mm ethidium bromide. The degradation of [3H]poly(C)-poly(I) by purified enzyme preparations yields labeled dinucleotides and octanucleotides; the latter oligonucleotide contained 5'-phosphate and 3'-hydroxyl termini. The enzyme cleaves high molecular weight RNAs such as RNA products formed in vitro by T3 phage-induced RNA polymerase from T3 phage DNA, heterogeneous RNA isolated from duck reticulocyte nuclei, and 45 S RNA isolated from rat liver nucleoli. The mode of degradation of RNA in vitro with the double-stranded RNase is similar to that of Escherichia coli RNase III and appears to act endonucleolytically. The degradation of 45 S RNA with the enzyme results in the production of 29 S and 19 S RNA fragments. These findings suggest that the enzyme may be involved in the processing of high molecular weight precursor RNAs to mRNA or rRNAs in a manner analogous to that reported for RNase III of E. coli.
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PMID:Isolation and purification of double-stranded ribonuclease from calf thymus. 83 40

Partially purified rat liver RNA polymerase I chromatographed on ribosomal RNA-Sepharose loses most (96%) of its activity assayed on native calf-thymus DNA templates, but loses little (8%) of its activity assayed on poly(deoxycytidylic acid) template. Polymerase I is not stimulated by polymerase II protein factor, or by bovine serum albumin. However, it is stimulated by histones, polylysine, and spermine. Addition of a protein fraction eluted by high ionic strength from the rRNA-Sepharose also restores activity on native calf-thymus DNA. Further purification yields a fraction containing two proteins of 11 000 and 12 000 molecular weight. Both proteins are distinct from histones by electrophoresis in sodium dodecyl sulfate and in acid urea. Both proteins are basic, insensitive to heat, bind to DNA, and stimulate polymerase I activity. The degree of stimulation of polymerase I is dependent upon both the enzyme/DNA and the factor/DNA ratio. The protein factors also stimulate polymerase II activity about half as effectively as polymerase I.
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PMID:A protein cofactor that stimulates the activity of DNA-dependent RNA polymerase I on double-stranded DNA. 85 26

Three forms of RNA polymerase were assayed in nuclei and nucleoli isolated from rat liver and from Krebs II ascites cells. Assays of rat liver nuclei in the absence of exogenous DNA showed polymerase I accounted for 72% of the total activity, polymerase II for 17%, and polymerase III for 11%. The total activity in ascites nuclei was similar but the ratios of polymerase activities were different: polymerase I, 53%; polymerase II, 41%; and polymerase III, 6%. These values may reflect differences in the transcriptional activity of the nuclei. After isolation of nucleoli, both rat liver and ascites polymerase I accounted for 85% of enzyme activity. When exogenous calf-thymus DNA was added to nucleoli, there was a greater than 50% increase in activity suggesting that less than one-half of the polymerase I present was bound to endogenous template. Polymerase I was solubilized from either rat liver or ascites nucleoli by sonication at high ionic strength and subsequently purified by ion filtration, phosphocellulose, sucrose gradient centrifugation, and DNA-cellulose chromatography. The essentially homogenous ascites enzyme had a specific activity of 86 units/mg when assayed with native calf-thymus DNA and of 876 units/mg when assayed with poly(deoxycytidylic acid). Electrophoresis of the enzyme in sodium dodecyl sulfate indicated the presence of six subunits with molecular weights of 205 000, 125 000, 51 000, 44 000, 26 000 and 16 000. After the same purification procedure, the rat liver enzyme had a similar specific activity (98 units/mg) on native calf thymus and 362 units/mg on poly(deoxycytidylic acid).
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PMID:Purification of rat liver and mouse ascites DNA-dependent RNA polymerase I. 85 54

A simplified method is described for the large-scale preparation of a highly purified DNA-dependent RNA polymerase B from calf thymus. The method includes homogenization and lysis of the tissue, chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite-Sephadex G-10 and, once again, phosphocellulose. The procedure avoids the preparation of nuclei, the use of sonication, ammonium sulphate precipitations and dialysis steps and needs no ultracentrifugation.
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PMID:An improved method for the preparation of the DNA-dependent RNA polymerase B from calf thymus. 87 42

The purpose of the present study was to determine the effects of covalent binding to DNA of a reactive derivative of benzo[a]pyrene on template activity during in vitro transcription with RNA polymerase. Calf thymus deoxyribonucleic acid, modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, was transcribed with Escherichia coli DNA-dependent RNA polymerase. With increasing levels of modification, there was a progressive inhibition of transcription. The inhibition was much greater under conditions where continuous reinitiation of transcription occurred than under conditions where only one RNA chain was synthesized per initiation site. This suggested that the modified sites block the movement of polymerase along the template and prevent recycling of the enzyme. Consistent with this interpretation were analyses of RNA transcripts on sucrose density gradients which showed a progressive decrease in average RNA chain length as the extent of template modification increased. In contrast to the inhibitory effect on chain elongation, evidence was obtained that the modified DNA had an increase in the number of initiation sites for transcription. These results are consistent with separate physical studies indicating that modification of DNA by this benzo[a]pyrene derivative can induce small localized regions of denaturation.
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PMID:Template activity of calf thymus DNA modified by a dihydrodiol epoxide derivative of benzo[a]pyrene. 88 93

HeLa cell interphase chromatin has been sheared and fractionated by isoelectric focusing. Chromatin fractions are obtained with a wide range of isoelectric points. No free DNA is observed. While protein/DNA rations are similar in the various fractions, they appear to contain different nonhistone chromosomal proteins. A minor chromatin fraction with isoelectric point congruent to 7.0 does not contain histone H1. This fraction is considerably more active as template with different RNA polymerases than the other fractions. Kinetic studies, in which RNA polymerase activity is assayed at various concentrations of chromatin, indicate that the greater activity of Escherichia coli RNA polymerase is due to an increased rate of transcription at saturating concentrations of template (Vmax) and is not due to a lower concentration required for half-maximal rate of transciption (Km). In contrast, the increased rate of transcription by calf-thymus RNA polymerases II and III is due to a decrease in chromatin concentration required for half-maximal rate of transcription rather than an increased rate of transcription at saturating concentrations of template. These results suggest that chromatin with isoelectric point congruent to 7 offers a greater frequency of binding sites for mammalian RNA polymerases, as would be expected for a "transcriptionally active" fraction.
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PMID:Composition and template activity of chromatin fractionated by isoelectric focusing. 91 91

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.
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PMID:Purification and properties of RNA polymerase I from Ehrlich ascites cells. 91 51

Crude calf thymus DNA-dependent RNA polymerase, RNA polymerase B (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), was incubated with the tritium labeled, potent inhibitor [3H]amanin, in order to form the enzymatically inactive [3H]amanin-polymerase complex ([3H]A-P complex). Subsequent purification procedures for the [3H]A-P complex were based on radioactive assays. Phosphocellulose chromatography separated two radioactive components: PCI, the previously reported amatoxin binding protein, ABP (Brodner and Wieland, 1976), and PC II, the [3H]A-P complex. Sodium dodecyl sulfate gel electrophoresis of the complex showed the presence of a new heavy band very close to subunit B 1 and a decreased intensity of subunit band B 3. These were the only differences noted in the subunit structure of RNA polymerase B. [3H]Amanin was covalently coupled to the enzyme, affinity labeling, by a water-soluble carbodiimide and the resultant conjugate submitted to sodium dodecyl sulfate gel electrophoresis. The profile of radioactivity showed one main peak (greater than 2000 cpm) coinciding with the 550-nm absorption peak of subunit B 3 on a stained parallel gel. Since no other protein band contains any significant radioactivity, the binding site for [3H]amanin and most probably for all amatoxins is localized on the B 3 subunit SB 3.
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PMID:Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B. 95 70

Two different forms of DNA-dependent RNA polymerase have been solubilized and purified from nuclei of Ehrlich ascites tumor cells. The purification procedure involves ammonium sulfate precipitation and gel filtration on Sephadex G-25. The separation of A and B activities is achieved by chromatography on DEAE-cellulose. Nuclei are prepared from cells, sensitive or resistant to daunorubicin. RNA polymerases A and B have an absolute requirement of divalent cations for activity. Native DNAs are better templates than heat-denatured DNAs for RNA polymerase A. On the contrary heat-denatured DNA is more transcribed than the native one by RNA polymerase B. The low level of transcription of total and nucleolar ascites DNAs is due to the DNA, the same results being obtained with ascites and calf thymus RNA polymerases A and B. The inhibitory action of daunorubicin on RNA polymerases A and B from Ehrlich ascites tumor cells has been studied in vitro. The same results are obtained with enzymes extracted from sensitive or resistant cells. Daunorubicin does not inhibit the binding of RNA polymerases to the DNA template, but prevents the transformation of the DNA-daunorubicin-RNA-polymerase unstable complex into the highly stable one. This inactive ternary complex has a dissociation rate faster than the stable complex formed without daunorubicin. The size of the RNA synthesized in the presence or absence of daunorubicin is the same.
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PMID:Daunorubicin inhibition of DNA-dependent RNA polymerases from Ehrlich ascites tumor cells. 99 57

Studies were carried out to elucidate the mechanism underlying the impairment in RNA synthesis in the rat thymus after whole-body exposure to X-rays (1000 rad). The ability of thymus nuclei to catalyse RNA polymerization in vitro was adversely affected in irradiated rats. Further experiments showed that whole-body irradiation caused a considerable decrease in both the activity of RNA polymerase and the template efficiency of chromatin but not in that of DNA isolated from chromatin. These radiation-induced changes in transcription seem to arise partly from the direct effect of radiation on the thymus itself and partly from the abscopal mechanism(s).
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PMID:Effect of whole-body irradiation on transcription in the rat thymus. 107 99


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