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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)

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) from Acinetobacter calcoaceticus was purified to apparent homogeneity and its properties were compared with those of the Escherichia coli B enzyme. The molecular weights of the two native active enzymes as well as their alpha and beta subunits appeared to be similar. No subunit corresponding to that of sigma from E. coli was found, and furthermore no separation between the beta subunits could be detected by gel electrophoresis. A number of different DNAs were transcribed by the enzyme from A. calcoaceticus. Maximal RNA synthesis occurred at pH 8.7, 10 mM Mg2+, or 0.3 mM Mn2+ and at a total ionic strength of 0.1. Higher ionic strengths led to increasing inhibition of transcription and at mu = 0.4 complete inhibition was observed. The mechanism of inhibition of salt was not related to the initiation event as observed with T4 core RNA polymerase (R.Kleppe, 1975). In an attempt to understand the mechanism of inhibition by salt, the effect of ionic strength on the sedimentation properties of the enzyme was investigated. At low ionic strength, enzyme species with sedimentation coefficients, s20,w, of 5.8S, 12.4S, and 19.3S were present. In buffers with higher ionic strengths the relative amounts of the 12.4S species decreased. It is suggested, therefore, that the inhibition of activity at higher salt concentrations is caused by a decrease in concentration of the active enzyme species.
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PMID:Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus. 0 80

A DNA-dependent RNA polymerase has been isolated from Caldariella acidophila, a thermophilic bacterium living in acidic hot springs at temperatures ranging from 63 to 89 degrees C. The enzyme was purified 180-fold and is composed of five different subunits having the following molecular weights: a = 127000, b = 120000, c = 72000, d = 65000, and e = 38000. The enzyme is activated by Mn2+ and Mg2+ and exhibits optimal activity in the presence of 0.5 mM Mn2+. The activity depends on ionic strength, with a maximum at 0.25 M KCl, and exhibits a pH optimum at 7.8 in the presence of Tris-HCl buffer. The enzyme shows a high degree of thermophilicity, its temperature optimum being 80 degrees C in the in vitro assay. The thermophilicity of C. acidophila RNA polymerase allows studies on enzyme-template interactions to be performed in a temperature range where many templates are close to their Tm.
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PMID:DNA-dependent RNA polymerase from the thermophilic bacterium Caldariella acidophila. Purification and basic properties of the enzyme. 0 9

The endogenous RNApolymerase activity of isolated cell nuclei and chloroplasts from young pea plants (Pisum sativum) has been studied. The presence of all four nucleoside triphosphates and Mg2+ ions is necessary for the reaction. The missing of one of these nucleotides from the reaction mixture, especially ATP, sharply decreases the transcription. Chloroplasts synthesize RNA per DNA unit more intensively, than nuclei. In spring such predominance is especially pronounced. Maximal synthesis of RNA is observed at pH 8.3 both in nuclei and chloroplasts. Maximal transcription was observed at 25 degrees in chloroplasts and at 30--35 degrees in nuclei. Actinomycin D inhibited the process of transcription both in nuclei and some stimulation in chloroplasts were observed, when rifamicin B was added. It is suggested that there are differences in nuclear and chloroplast forms of RNA polymerase.
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PMID:[Comparative study of endogenous RNA-polymerase activity of the cell nuclei and chloroplasts of pea leaves]. 0 71

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

Endogenous Mn2+ and Mg2+ activated RNA polymerase activity was measured in isolated cell nuclei of brain from rats of different age-groups. It was established that the activity of the nucleoplasmic RNA polymerase is maintained at the level of young animals up to an age of 16 months but is decreased after 24 months. The nucleolar RNA polymerase activity decreases already at 16 months and a higher ratio of the Mn2+:Mg2+ activated RNA polymerase activities has been found to be characteristic for the older animals. By means of stepwise sucrose density gradient centrifugation fractions were obtained from the nuclear preparations highly enriched in cell nuclei of neuronal and glial origin respectively. By measuring the activity of the nucleoplasmic and nucleolar RNA polymerases in these fractions it was found that the elevated ratio of the nucleoplasmic to nucleolar RNA polymerase activity at 16 months of age is a characteristic of the neuronal nuclei while the glial nuclei behave by the opposite manner. A parallelism existing between the age-dependent change of the endogenous RNA polymerase activity and that of perichromatic granules of rat brain cortical cells is discussed.
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PMID:Age-dependent alterations of the rate of RNA synthesis in rat brain cell nuclei. 1 35

Rabies virion-associated transcriptase activity was investigated in vitro and compared with that of the New Jersey serotype of vesicular stomatitis virus. The concentration of detergent that affected [3H]GMP incoporation into acid-insoluble material was significantly different for both viruses. Vesicular stomatitis virus New Jersey required 0.05 to 0.1% nonionic detergent, whereas rabies virion could not be fully activated unless 4 to 5% detergent was used. Other optimal conditions were as follows: 40 mM NaCl, 5 mM Mg2+, 40 mM Tris-hydrochloride (pH 7.4), 5 mM dithiothreitol, and 30 degrees C. The reaction required four nucleoside triphosphates. The initial rate of RNA synthesis by rabies virion enzyme was 140 pmol of GMP incorporated/mg of viral protein per h and linearly increased until about 8 h, with a slight initial lag phase. The enzyme activity that correlated with the content of L protein was highest when rabies virions were grown at 33 degrees C. The product was single-stranded RNA, which was complementary in base sequences to rabies viral RNA. Most of the RNA synthesized sedimented at 6-16S.
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PMID:Transcriptase activity associated with rabies virion. 2 66

Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis.
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PMID:Inhibitory effects of nucleoside triphosphates on nucleolar RNA synthesis. 3 1

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

An adenosinetriphosphatase (ATPase) [EC 3.6.1.3] copurified with the DNA-dependent RNA polymerase [EC 2.7.7.6] from Escherichia coli was isolated to apparent homogeneity and some of its functional as well as structural properties were examined. Although the novel ATPase exhibited metal requirements similar to those of Mg2+, Ca2+-ATPase, its response to NaN3 and antisera appeared completely different from that of the Mg2+, Ca2+-ATPase. The purified ATPase was found to be a large protein with a molecular weight of 9.3X10(5) daltons, composed of identical subunits of 7X10(4) daltons. When viewed under an electron microscope, the ATPase appeared to be very similar to material previously misidentified as the RNA polymerase. The physiological role of the novel ATPase, however, remains unclear.
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PMID:A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli. II. Enzymatic properties and molecular structure. 13 30

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.
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PMID:Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition. 14 Jan 66


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