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)

phi 227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 X 10(10) to 2 X 10(10) PFU/ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of phi227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed ty hydrochloric acid, trichloracetic acid, and hot formamide: however, formamide-extracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 43 degrees C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that phi227 does not code for a rifampin-resistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased suggesting that altered host core polymerase had less affinity for (some) promotors on the phi227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 X 10(6) and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available.
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PMID:Characterization of group H streptococcal temperate bacteriophage phi 227. 1 33

RNA polymerase was extracted from the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV)-induced C3H/He mouse ascites sarcoma cells (SR-C3H). RNA polymerase was separated into RNA polymerases I and II by DEAE-Sephadex chromatography. RNA polymerase I was separated into Ia and Ib fractions by phospho-cellulose chromatography. In SR-C3H cells RNA polymerase Ib was the main component of RNA polymerase I. At 0.05--0.1 M ammonium sulphate RNA polymerase I transcribed native DNA most actively, and RNA polymerase II transcribed denatured DNA most actively. Partial digestion of DNA by DNAase I enhanced RNA synthesis by RNA polymerases I and II. At ionic strength over 0.2 M ammonium sulphate, the initiation reaction of RNA polymerases I and II was inhibited. The initiation complexes of RNA polymerases I and II with native DNA were more stable against high salt concentration than with denatured DNA.
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PMID:Characterization of RNA polymerases from Rous sarcoma virus-induced mouse ascites sarcoma cells. 3 35

Two forms of DNA-dependent RNA polymerase have been partially purified (about 100-fold relative to the crude extract) from 48-h old cells of Streptomyces antibioticus. The two forms show different Mg2+ optima for the incorporation of [3H]UMP into RNA. Substances inhibiting transcription have been isolated by ammonium sulfate precipitation from one of the fractions produced during the polymerase purification. Actinomycin can be shown to inhibit RNA synthesis catalyzed by the S. antibioticus polymerases to a similar extent regardless of the template used. When S. antibioticus DNA is the template, actinomycin inhibits transcription by S. antibioticus polymerase to a degree that is significantly less than the observed actinomycin inhibition of synthesis catalyzed by Escherichia coli polymerase or by either S. antibioticus or E. coli polymerase with calf thymus DNA as the template. Using an assay previously developed, it was shown that the association constant for the binding of actinomycin to S. antibioticus DNA was increased by the presence of RNA polymerase in the binding mixture, while the association constant for the binding to calf thymus DNA was decreased by RNA polymerase. RNA synthesis in crude, cell-free extracts of 12-h old S. antibioticus cells (not producing actinomycin) is less refractory to actinomycin inhibition than synthesis catalyzed by extracts of 48-h old (actinomycin producing) cells, and both extracts catalyze appreciable RNA synthesis at actinomycin concentrations that completely inhibit RNA synthesis catalyzed by E. coli extracts.
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PMID:RNA synthesis in Streptomyces antibioticus: in vitro effects of actinomycin and transcriptional inhibitors from 48-h cells. 6 Jan 28

DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.
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PMID:Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta. 10 73

The DNA polymerase of early embryos of Drosophila melanogaster has been purified to near-homogeneity. The purified enzyme gave a single, catalytically active protein band after polyacrylamide gel electrophoresis, under nondenaturing conditions. Four polypeptides with molecular weights 43,000, 46,000, 58,000, and 148,000 were resolved when this band was electrophoresed under denaturing conditions. At high ionic strengths, the DNA polymerase had a sedimentation coefficient of 8.7 S, a Stokes radius of 78 A and frictional ratio of 1.81, parameters that yield a molecular weight of 280,000. The purified DNA polymerase possessed no detectable endo- or exodeoxyribonuclease, ATPase, or RNA polymerase activity. Using an "activated" DNA template-primer, the enzyme had a pH optimum of 8.5. It was stimulated by (NH4)2SO4, KCl, and to a lesser extent, NaCl. A divalent metal cation was absolutely required; MgCl2 stimulating activity 7-fold more than MnCl2. It was inhibited by low concentrations of N-ethylmaleimide and Aphidicolon. Thus the DNA polymerase of D. melanogaster resembles most closely the alpha-DNA polymerases that have been purified from mammalian cells.
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PMID:A high molecular weight DNA polymerase from Drosophila melanogaster embryos. Purification, structure, and partial characterization. 11 15

Correct termination of transcription of the rRNA gene in nucleoli isolated from Tetrahymena is dependent on a protein factor with a molecular weight between 50,000 and 100,000. At low ionic strength the endogenous RNA polymerase synthesizes a transcript identical in size to the precursor rRNA extracted from cells. In the presence of ammonium sulfate, however, the RNA polymerase reads through the normal termination point as demonstrated by size and hybridization studies of the transcript. After ammonium sulfate treatment, rDNA associated with chromosomal proteins (r-chromatin) can be separated from the termination factor by differential centrifugation. The endogenous RNA polymerase on the salt-treated r-chromatin is no longer able to recognize the normal terminator even at low ionic strength. Normal termination properties can be reconstituted by complementation with intact nucleoli or with a protein factor extracted from nucleoli.
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PMID:Termination of transcription in nucleoli isolated from Tetrahymena. 11 52

In the presence of 50 mM (NH4)2SO4 and low concentrations of alpha-amanitin (7.7 mug/ml), adrenal nuclei synthesize predominately rRNA as characterized by size and base composition. Approximately 10% of the RNA synthesized under these conditions sediments at 4-5 S; this RNA synthesizing activity is inhibited by high concentrations of alpha-amanitin (231 mug/ml) indicating the presence of RNA polymerase III activity. ACTH administration to guinea pigs results in a twofold increase in adrenal nuclear RNA polymerase I and III activities at 14 hr of hormone treatment. Analysis of the amount of radiolabeled nucleoside triphosphate incorporated in vitro into 3' chain termini and into internal nucleotide positions has been utilized to measure the number of RNA chains and the average chain length synthesized in vitro. Incorporation into 3' chain termini is not changed by ACTH; incorporation into internal nucleotides is doubled in parallel with the increase in RNA polymerase I activity. These results are not due to an altered Km of RNA polymerase I for the four nucleoside triphosphates, nor to differential R Nase or phosphatase activity. These studies suggest that the regulation of RNA polymerase I by ACTH is accomplished in part through an increase in the rate of RNA chain elongation.
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PMID:Adrenocorticotropic hormone stimulation of adrenal RNA polymerase I and III activities. Nucleotide incorporation into internal positions and 3' chain termini. 16 11

DNA-dependent RNA polymerase (EC 2.7.7.6) ACTIVITIES FROM NORMAL BHK-21/C13 cells and from BHK-21/C13 cells transformed by polyoma virus (PYY cells) were solubilized and fractionated on columns of DEAE-Sephadex. Various properties of the A and B enzymes from the two types of cell were compared. 1. The yields of polymerase relative to the DNA content of the nuclear preparations are similar for both cell types. 2. The ionic-strength optima of polymerases A and B are 12.5 mM and 100mM with respect to (NH4)2SO4 for both cell types. 3. The Mn2+/Mg2+ activity ratio (measured at the respective optimum for each cation) for polymerase A from BHK-21/C13 cells was 1.48 and for the polymerase A from PYY cells was 0.55. The corresponding ratios for polymerase B were 10.11 for BHK-21/C13 cells and 22.75 for PYY cells. 4. Minor differences in the ability of the A polymerases to transcribe native and denatured DNA templates were observed; such differences were not apparent when the B polymerases were compared. 5. All the polymerases were inhibited completely by actinomycin D and by rifampicin AF/013, but not markedly so by rifampicin. Alpha-amanitin inhibited polymerase B but not polymerase A.
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PMID:Deoxyribonucleic acid-dependent ribonucleic acid polymerases from normal and polyoma-transformed BHK-21/C13 cells. 16 71

DNA-dependent RNA polymerases were extracted from the nuclei of poorly differentiated tumor, Morris hepatoma 3924A, and purified by an initial chromatography on a DEAE-Sephadex column followed by fractionation on phosphocellulose and finally on a second DEAE-Sephadex column. Three major forms of RNA polymerase (IA, IB and II) were resolved chromatographically. Enzymes IA, IB and II eluted from DEAE-Sephadex at 75, 150 and 210 mM (NH4)2SO4, respectively. The specific activities (nmol UMP incorporated mg protein per 15 min) of polymerases IA, IB and II were 40, 43 and 182, respectively. Concurrently, DNA-dependent RNA polymerases were extracted from normal liver and subjected to similar chromatographic procedure. Upon the final DEAE-Sephadex chromatography, enzymes IA, IB and II eluted at 110, 180 and 210 mM (NH4)2SO4, respectively. The recovery of polymerases IA, IB and II after purification was 0.21, 0,28 and 0.42 unit/mg DNA, respectively, for hepatoma enzymes and 0.07, 0.05 and 0.42 unit/mg DNA for the corresponding liver enzymes.
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PMID:RNA polymerases from a rat hepatoma. Partial purification and comparison of properties with corresponding liver enzymes. 17 77

The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.
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PMID:Stimulation of ascites tumor RNA polymerase II by protein kinase. 17 56


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