Purification of rat liver and mouse ascites DNA-dependent RNA polymerase I.
Three forms of RNA polymerase were assayed in nuclei and nucleoli isolated from rat liver and from Krebs II ascites cells. Assays of rat liver nuclei in the absence of exogenous DNA showed polymerase I accounted for 72% of the total activity, polymerase II for 17%, and polymerase III for 11%. The total activity in ascites nuclei was similar but the ratios of polymerase activities were different: polymerase I, 53%; polymerase II, 41%; and polymerase III, 6%. These values may reflect differences in the transcriptional activity of the nuclei. After isolation of nucleoli, both rat liver and ascites polymerase I accounted for 85% of enzyme activity. When exogenous calf-thymus DNA was added to nucleoli, there was a greater than 50% increase in activity suggesting that less than one-half of the polymerase I present was bound to endogenous template. Polymerase I was solubilized from either rat liver or ascites nucleoli by sonication at high ionic strength and subsequently purified by ion filtration, phosphocellulose, sucrose gradient centrifugation, and DNA-cellulose chromatography. The essentially homogenous ascites enzyme had a specific activity of 86 units/mg when assayed with native calf-thymus DNA and of 876 units/mg when assayed with poly(deoxycytidylic acid). Electrophoresis of the enzyme in sodium dodecyl sulfate indicated the presence of six subunits with molecular weights of 205 000, 125 000, 51 000, 44 000, 26 000 and 16 000. After the same purification procedure, the rat liver enzyme had a similar specific activity (98 units/mg) on native calf thymus and 362 units/mg on poly(deoxycytidylic acid).
D000818:Animals; D002287:Carcinoma, Krebs 2 / Q000201:enzymology*; D002458:Cell Fractionation; D002466:Cell Nucleolus / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D002846:Chromatography, Affinity; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D012318:RNA Polymerase I / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013010:Sonication; D013698:Templates, Genetic
D046911:Macromolecular Substances; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I