Nucleolar DNA-dependent RNA polymerase from rat liver. 1. Purification and subunit structure.
DNA-dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE-Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electrophoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%. The RNA polymerase I eluted as a single peak from DEAE-Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20% of the total RNA polymerase I activity and the IB enzyme comprised approximately 80%. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130000 (b), 65000 (c), 40000 (d), and 19000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme. Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme.
D000818:Animals; D002466:Cell Nucleolus / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D012318:RNA Polymerase I / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I