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)

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.
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PMID:Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. 37 77

An enzyme has been isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages phi X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dna B-dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically with DNA polymerases III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drd11 or ColIdrd1, which are derepressed in functions of conjugational DNA transfer, severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis.
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PMID:A DNA primase specified by I-like plasmids. 38 43

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.
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PMID:Identification of two different RNase H activities associated with yeast RNA polymerase A. 38 60

The molecular structure of RNA polymerases from Escherichia coli, Salmonella typhimurium, Salmonella anatum,serratia marcescens, Aerobacter aerogens, Proteus mirabilis and Bacillus subtilis were compared based on:i) inhibition of the enzyme activity by treatment with antibodies against E. coli RNA polymerase subunits;ii) analysis of antibody precipitates by sodium ododecyl sulfatepolyacrylamide gel electrophoresis; and iii) analysis of antibody precipitates by urea-isoelectrofocusing followed by sodium dodecyl sulfate-slab gel electrophoresis in the second dimension. All the bacterial RNA polymerases examined cross-react equally with anti-E. COLI HOLOPOLYMERASE BUT EXHIbit different extents of cross-reaction with antibodies against individual subunits. Except for B. subtilis RNA polymerase, the molecular weight and isoelectric point of the enzyme subunits are close to those of E. coli polymerase. However, minor difference were found at least within the resolution of the techniques employed:S. anatum polymerase has sigma subunit larger than E. coli sigma subunit; P. mirabilis enzyme has sigma subunit larger in size and more acidic in charge, and alpha subunit smaller and more basic than corresponding E. coli subunits. The electrophoretic map of B. subtilis enzyme subunits is completely different from that of E. coli enzyme.
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PMID:Comparative studies of RNA polymerase subunits from various bacteria. 40

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepharose column which bound the beta' subunit. A phosphocellulose column was used to separate the alpha and beta subunits. During purification, the enzyme eluted from the DNA-cellulose column in three separate forms in the order alpha2betabeta'deltaomega1,alpha2betabeta'omega1, and alpha2betabeta'omega1sigma. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as beta. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase.
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PMID:Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase. 41 92

A heat-stable protein (HSF) that stimulates the activity of lamb thymus RNA polymerase II has been purified 2500-fold and partially characterized. This factor stimulates the activity of RNA polymerase II up to 13 times and retains complete activity when heated at 90 degrees C for 5 min. Stimulation is observed only in the presence of RNA polymerase II and requires native DNA as template. The stimulatory factor has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 9.55 x 10(-7) cm2/s, and an isoelectric point of 8.0. Calculated from the sedimentation and diffusion data, the factor has a molecular weight of about 24,000. Electrophoresis of the purified factor on polyacrylamide gels in the presence of sodium dodecyl sulfate results in a single band corresponding to a molecular weight of 25,000. The number-average length of the RNA synthesized by RNA polymerase II is increased in the presence of the factor. Sedimentation velocity and exclusion chromatography experiments suggest that the stimulatory factor interacts with RNA polymerase II. These results suggest that the factor stimulates RNA synthesis through a direct interaction with RNA polymerase II. The stoichiometry of the HSF-RNA polymerase binding appears to be about 1:1. HSF is located in the nucleus, as determined by cell fractionation studies.
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PMID:Purification and partial characterization of a stimulatory factor for lamb thymus RNA polymerase II. 43 87

Rapid and reliable fractionation of neuronal and nonastrocytic glial (NAG) cerebral rat brain chromatin in transcribable and repressed portions was achieved employing the DNAase II/Mg++-solubility method of Gottesfeld et al. (1974). Compositional and transcriptional properties of these fractions have been investigated. Compared to transcriptionally repressed fractions, template-active neuronal and NAG chromatin fractions are associated with an increased content of nonhistone chromosomal (NHC-) proteins. Both of the transcribable as well as both of the repressed fractions are strikingly different in their composition as assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Comparative acid urea gel electrophoretic patterns of histones revealed that histone fraction H 1 is almost completely absent in actively transcribed neuronal chromatin and reduced in the corresponding NAG fraction while in template-inactive neuronal and NAG chromatin all five main histone fractions are present in equal amounts. The total number of RNA initiation sites available for exogenously added homologous RNA polymerase on template-active and -inactive neuronal and NAG chromatin was quantitatively measured under assay conditions completely eliminating reinitiation. Unlike the template-active neuronal and NAG fractions which are differently enriched in RNA initiation sites, transcriptionally more repressed neuronal and NAG fractions demonstrated a minimal ability to initiate RNA synthesis. Under assay conditions allowing repeated initiation of RNA chains at the same initiation site, rat brain RNA polymerase molecules were found to utilize neuronal initiation sites more frequently than NAG ones.
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PMID:Characteristics of transcriptionally active and inactive neuronal and nonastrocytic glial rat brain chromatin fractions. 43 84

RNA polymerase II polypeptides present in [35S]methionine-labeled Chinese hamster ovary (CHO) cell extracts have been quantitatively immunoprecipitated with an anti-calf thymus RNA polymerase II serum. Analyses of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels indicated that the immunoprecipitated polymerase II of both wild type CHO cells and the alpha-amanitin-resistant mutant Ama1 had polypeptides of molecular weight 214,000, 140,000, 34,000, 25,000, 23,000, 20,500, and 16,500. In heterozygous alpha-amanitin-resistant/alpha-amanitin-sensitive hybrid CHO cells, growth in the presence of alpha-amanitin results in the inactivation of the alpha-amanitin-sensitive RNA polymerase II activity and a compensating increase in the activity of the alpha-amanitin-resistant enzyme. Determination of the rates of synthesis and degradation of RNA polymerase II polypeptides using [35S]methionine labeling and polymerase II immunoprecipitation demonstrated that this increase in activity of alpha-amanitin-resistant polymerase II resulted from a co-ordinate increase in the rate of synthesis of at least three polypeptides of RNA polymerase II. At the same time, there was an enhanced rate of degradation of the alpha-amanitin-inactivated RNA polymerase II polypeptides.
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PMID:Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines. 43 82

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


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