ABSTRACT
DNA-Dependent RNA polymerase (EC 2.7.7.6) was isolated from Thermomonospora curvata. The purification steps included precipitation with Polymin P, elution of the precipitate with 0.3 mol/L KCl, precipitation with ammonium sulfate, affinity chromatography on heparin-agarose and molecular filtration on Biogel A 1.5 m.
Subject(s)
Actinomycetales/enzymology , Bacterial Proteins/isolation & purification , DNA-Directed RNA Polymerases/isolation & purification , Chemical Precipitation , Chromatography, Affinity , Chromatography, Gel , Molecular Weight , Spores, BacterialABSTRACT
DNA-directed RNA polymerase was isolated from wild-type and mutant (asporogenic and granaticin overproducing) Streptomyces granaticolor. The effect of granaticin on the enzyme activity was compared with that of standard transcription inhibitors, rifampicin and rifamycin SV and shown to be zero.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Streptomyces/enzymology , Anti-Bacterial Agents/pharmacology , Naphthoquinones/pharmacology , Streptomyces/drug effectsABSTRACT
RNA nucleotidyltransferase from Streptomyces granaticolor was purified and some of its properties were investigated. The temperature optimum of the enzyme is 35 degrees C, pH optimum 7.8-8.4. Antibodies against the beta subunit of the enzyme from Bacillus subtilis immunologically cross-react with the beta subunit of the enzyme from S. granaticolor. Antibodies against the beta' subunit of the enzyme from B. subtilis immunologically cross-react with both beta and beta' subunits of the enzyme from S. granaticolor.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Streptomyces/enzymology , Bacillus subtilis/enzymology , Cross Reactions , Hydrogen-Ion Concentration , Immune Sera , Kinetics , ThermodynamicsABSTRACT
RNA nucleotidyltransferase (EC 2.7.7.6) of Streptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA- cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be beta and beta subunits, respectively. The role of other subunits of the enzyme is discussed.
Subject(s)
DNA-Directed RNA Polymerases/isolation & purification , Streptomyces/enzymology , Bacterial Proteins/analysis , Chemical Precipitation , Chromatography, Affinity , Chromatography, Gel , DNA-Directed RNA Polymerases/analysis , Electrophoresis, Polyacrylamide GelABSTRACT
An auxotrophic strain of Escherichia coli with the recB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1 drd19). The donor plasmid DNA obtained by cell lysis in the presence of Triton X-100 and subsequent centrifugation in a caesium chloride-ethidium bromide gradient was shown to have a circular molecule and to retain its completeness after penetration into the recipient. Experiments with mixtures or plasmid and chromosomal DNA indicate a competition between these two DNA types during the transformation reaction in the given system.
