DNA-dependent RNA polymerase I from human placental nuclei

This paper describes a large-scale method for solubilisation and purification of DNA-dependent RNA-Polymerase I from mature human placenta. The solubilisation method involves homogenization of the whole human placenta, isolation of cell nuclei, sonication of separated nuclei at high ionic strength and ammonium sulfate precipitation. The purification method consists of chromatography of RNA-Polymerase I activity on DEAE-Sephadex A-25 and Phosphocellulose P-11, and glycerol-density gradient centrifugation. In result, RNA-Polymerase I of human placenta nuclei has been shown to be completely resistant to alpha-amanitin. Besides dependence of RNA-Polymerase I on different Mg2+ and Mn2+ concentrations, glycerol concentration and ionic strength was studied. Using our results, an optimal RNA-Polymerase I assay mixture was developed. The subunit composition of RNA-Polymerase I was investigated by dodecylsulfate-gel electrophoresis. The RNA-Polymerase I molecule of human placenta consists of 13-14 polypeptides.     

A novel factor for DNA-dependent RNA polymerase II in rat liver nuclei

The author found, in rat liver nuclei, a novel factor which exhibited a strong inhibitory effect on RNA chain initiation by various classes of RNA polymerases (I, II and III) using an exogenous DNA template.The molecular weight of this factor was estimated to be 70 K daltons, and its activity was not affected by treatment with trypsin, RNase A, lipase C, -amylase and heat. However, its activity was inactivated by a digestion of glycosidases. The molecule is shown to contain a considerable amount of sugars by physicochemical analysis. In addition, it is elucidated that the factor is not heparin which has a similar biological activity.     

Isolation and characterization of DNA-dependent RNA polymerase I of Tetrahymena pyriformis

A procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2.7.7.6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase I was identified by its resistance against alpha-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180,000, 118,000, 37,500, 36,000, 29,000, 27,500, 20,000, 18,500, 15,600, 14,500, 13,500, and 12,600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R.G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285-329, Cold Spring Harbor Publ. Co., New York].     

DNA-dependent RNA polymerase II from nuclei of suspension-cultured tobacco cells

From isolated nuclei of suspension cultured cells of Nicotiana tabacum. DNA-dependent RNA polymerase II (E.C. 2.7.76) has been purified to homogeneity as evidenced by polyacrylamidegel electrophoresis under non-denaturing conditions. The purified enzyme had a specific activity of more than 15 nmol min^-1·mg^-1 with denatured calf thymus DNA as template. Sodium-dodecyl-sulfate gel electrophoresis and protein highperformance liquid chromatography revealed a subunit composition of four proteins with molecular weights of 165 000, 135 000, 35 000 and 25 000 and with a stoichiometry of 1:1:2:2. The RNA polymerase did not exhibit any detectable proteinkinase activity. The 25 000 subunit binds ADP in a molar ratio of 1:1; it could not be decided whether this subunit has an ATPase activity or is merely an acceptor of ADP.Abbreviations HPLC high-performance liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate This contribution is dedicated to Professor Fritz Cramer on the occasion of his 60th birthday     

Isolation of DNA-dependent RNA polymerase from the thermophilic actinomyceteThermomonospora curvata

DNA-Dependent RNA polymerase (EC 2.7.7.6) was isolated fromThermomonospora 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 1.5m.     

Purification and partial characterization of DNA-dependent RNA polymerase from Rhodomicrobium vannielii

DNA-dependent RNA polymerase has been isolated from Rhodomicrobium vannielii. Like those from other eubacteria, the enzyme contained four subunits: beta and beta prime (Mr 155,000), alpha (Mr 38,000), and sigma (Mr 98,000). Analysis by isoelectric focussing showed that both alpha and sigma had several forms with different isoelectric pH values. The enzyme was sensitive to rifampicin (5 ng rifampicin ml-1 gave 50% inhibition) and capable of specific promoter selection with DNA from R. vannielii, calf thymus and phage T7D111.     

DNA-dependent RNA polymerase from the nuclei of the spleen of white rats]

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.     

Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum.

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.     

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.     

Isolation and characterization of DNA-dependent RNA polymerases from rabbit mammary gland

RNA polymerases AI, AII, BI, BII, CI and CII were found in the mammary gland from lactating rabbits. The enzymes obtained from total cell homogenates were partially purified and separated by DEAE-Sephadex chromatography. Their chromatographic properties, alpha-amanitin-sensitivity, template specificity, ionic strength and divalent cation requirements are described.