Properties and subunit composition of RNA polymerase II from plant cell cultures.
The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.
D000546:Amanitins / Q000494:pharmacology; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013698:Templates, Genetic
D000546:Amanitins; D046911:Macromolecular Substances; D008345:Manganese; D012321:DNA-Directed RNA Polymerases