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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)

An improved method is described for the purification of the DNA-dependent RNA polymerase [ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6] from Escherichia coli. The method involves lysozyme-sodium deoxycholate lysis, low-speed centrifugation, precipitation with Polymin P, elution from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DNA-cellulose and Bio-Gel A 5m. RNA polymerase is purified to electrophoretic homogeneity in 2 days with a recovery of 45%, resulting in a yield of 250 mg of holoenzyme from 500 g of cells.
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PMID:A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. 110 52

Three forms of DNA-dependent RNA polymerase have been separated by chromatography of extracts of yeast-like cells and mycelium of the dimorphic fungus Mucor rouxii. Each of the three eznymes has been purified by means of protamine sulfate precipitation, ion exchange chromatography, affinity chromatography, and velocity sedimentation. Electrophoresis under denaturing conditions showed differences in the subunit compositions of all three purified enzymes. The properties of the enzymes from M. rouxii were similar to those of polymerases from other eukaryotic organisms. Denatured DNA was a better template than native DNA for all three enzymes but each enzyme had a distinct pattern of activities with different templates. Enzymes I and III displayed optimal activity with Mn-2gs the divalent cation and were stimulated significantly by Kcl and (NH4)2S04. Enzyme II had a greater activity with Mg-2gnd was only slightly stimulated by KCl and (NH4)2SO4. None of the enzymes were inhibited by cycloheximide or by rifampicin: all were inhibited by actinomycin C and rifampin AF/018: only enzyme II was inhibited by alpha-amanitin. No differences could be found in the properties of the same enzymes isolated from yeast-like cells or mycelium.
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PMID:Deoxyribonucleic acid-dependent ribonucleic acid polymerases in the dimorphic fungus Mucor rouxii. 111 77

The DNA-dependent RNA polymerase from Pseudomonas BAL-31, the host for bacteriophage PM2, has been purified 154-fold using differential centrifugation, chromatography on DEAE-cellulose, ammonium sulfate precipitation, and sucrose gradient centrifugations at low and high ionic strength. The resulting enzyme is free of enzyme activities which could interfere with transcription studies and is greater than 85% pure as judged by polyacrylamide gel electrophoresis. Like other bacterial RNA polymerases, its subunit structure is beta'beta sigma alpha2. From gel electrophoresis the beta', beta, and alpha subunits have approximately the same molecular weights as those from Escherichia coli, whereas the sigma subunit is 5% larger (89,000 vs. 85,000). A summation of the subunits yields a molecular weight of 485,000 for the holoenzyme. Like other bacterial RNA polymerases, it sediments as a monomer (15 S) at low ionic strength (0.065) and as a dimer (22 S) at high ionic strength (0.75). Its activity is stimulated three-fold by monovalent cations (K+,NH4+, NA+) with additional stimulation provided by divalent cations (Mg2plus, Mn2plus). The transcription of phage PM2 form I (supercoiled) DNA has an ionic strength optimum of 0.26 for continuous long-term synthesis, and over an ionic strength range of 0.09-0.46 "plateau-type" kinetics are not observed. The sigma subunit is required for optimal PM2 transcription. The enzyme is sensitive to the same inhibitors of transcription as the RNA polymerase from E. coli, it has a temperature optimum of 28 degrees, and it is 50% inactivated by heating 10 min at 41 degrees. It has template preference similar to E. coli polymerase and shows little preference for homologous templates. With various DNAs the order of template activities is T7 greater than PM2 I congruent to T4 greater than PM2 II (relaxed circular form) greater than lambda-c greater than calf thymus greater than BAL-31 DNA. Phage PM2 form I DNA is transcribed at a twofold greater rate than PM2 form II DNA by this enzyme.
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PMID:DNA-dependent RNA polymerase from Pseudomonas BAL-31. I. Purification and properties of the enzyme. 112 Jan 4

DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).
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PMID:Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III. 112 92

A procedure has been developed for the purification of soluble DNA-dependent RNA polymerase (EC 2.7.7.6) from rye embryos. The enzyme solubilized by high salt extraction with sonication and resolved by DEAE-cellulose chromatography yields two activities. Enzyme I eluted at 0.15 M (NN4)2SO4, was insensitive to alpha-amanitin and was extremely labile. Enzyme II eluted at 0.25 M (NH4)2SO4 was inhibited by alpha-amanitin. However, DEAE-Sephadex chromatography yields three DNA-dependent RNA polymerases. Enzyme I is resistant to amanitin, while II and III enzymes are inhibited by this poison. Partially purified on DEAE-cellulose, polymerase II was further purified by hydrophobic chromatography on an omega-aminobutyl-Sepharose column. After omega-aminobutyl-Sepharose chromatography, enzyme II was stable and was more active with denatured than with native DNA as template. The activity of purified RNA polymerase II is dependent on the DNA, Mn-2+ and Mg-2+ added and requires ATP, GTP, CTP and UTP for its maximum activity. Transcription is inhibited besides by alpha-amanitin, by chromomycin A3, daunomycin, ethidium bromide and actinomycin D. Rifampin and rifamycin SV do not inhibit the enzyme. Synthetic copolymers were also effective as templates.
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PMID:Isolation and purification of RNA polymerases from rye embryos. 112 11

DNA-dependent RNA polymerase was isolated from rat spleen cell nuclei and was identified as A and B RNA polymerases by data on DEAE- and P-cellulose ionic exchange chromatography and on concentration dependency on bivalent ions and (NH4)2SO4. Two forms of the enzyme differed from each other in the activity in RNA synthesizing system, and their activity was completely inhibited by actinomycin, DNase and RNase.
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PMID:[DNA-dependent RNA polymerase from the nuclei of the spleen of white rats]. 113 3

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.
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PMID:Properties and subunit composition of RNA polymerase II from plant cell cultures. 114 41

Both forms A and B RNA polymerases solubilised from rat liver nuclei transcribed templates within these organelles when added exogenously to freshly prepared nuclei. The enzymes initiated more efficiently in the presence of KCL than ammonium sulphate and required manganese rather than magnesium as the divalent cation. Form A enzyme initiated most successfully at 375 mM KC6, activity was proportional to the amount of template added and continued linearly for at least 30 min. Form B enzyme initiated with two ionic strength optima, 125 mM and 500 mM KCl. Activity in the latter case was critically dependent on the enzyme: nuclei ratio. In both instances incorporation of nucleotide precurors was linear for less than 20 min. Form A enzyme synthesised products with a size distribution mainly larger than 18 S; form B enzyme synthesised products of mainly less than 5 S at 125 mM KCl and about 10 S at 500 mM KCl. Subfractionation of nuclei indicated that exogenous RNA polymerase A activity and form B at 125 mM KCl were occurring in nucleoli; form B activity at 500 mM KCl was nucleoplasmic. Measurements of U : G ratios in the RNA products suggested that exogenous form A was synthesising species with similar base ratios to the ribosomal RNA precurosrs. Both enzymes formed rifamycin AF/0-13 resistant complexes with nucleolar templates. Size analyses of products showed that whereas form B enzyme synthesised very small RNA species, RNA polymerase A produced a range of species of similar sizes to the ribosomal RNA precurosors.
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PMID:Transcription of isolated nuclei and nucleoli by exogenous RNA polymerase A and B. 114 40

Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from alpha-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.
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PMID:Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in rat liver after protein restriction. 115

Bovine lymphosarcoma tissue has been extracted with low- and high-salt buffers [0.05 M Tris-C1 plus or minus 0.3 M (NH4) 2S04]. Diethylaminoethyl-Sephadex chromatography of both the high-salt and low-salt extracts yields RNA polymerases I and II, although low-salt extraction releases only one-third as much activity. Extraction by high salt of the residue from the low-salt extract, followed by diethylaminoethyl-Sephadex chromatography, yields additional enzyme activity with properties of Form II. Purification of the low-salt extract by protamine precipitation, elution with sodium succinate, and phosphocellulose chromatography yields a preparation of RNA polymerase (RNAP) with hybrid properties, combining the salt optimum of Form I, diethylaminoethyl-Sephadex elution pattern of form II, and alpha-amanitin sensitivity of Form III. RNAP. transcribes native D,A and chromatin efficiently. More RNAPL is recovered from lymphosarcoma tissue than from calf thymus.
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PMID:RNA polymerase isolated from bovine lymphosarcoma by sequential low- and high-salt extraction. 117 19


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