Purification and subunit structure of RNA polymerase II from the pea

DNA-dependent RNA polymerase II (EC 2.7.7.6) from pea seedlings (Pisum sativum var. Alaska) has been purified to homogeneity, as judged by native polyacrylamide electrophoresis. The procedure includes polyethyleneimine precipitation and elution, ammonium sulfate precipitation, DEAE-Sephadex chromatography, phosphocellulose chromatography, and heparin-Sepharose chromatography. The enzyme purified almost to homogeneity has a specific activity of 200 nmol/mg per 15 min at 30 degrees C with denatured calf thymus DNA as template. The enzyme activity is 50% inhibited in the presence of 0.05 migrograms/ml of alpha-amanitin. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that pea RNA polymerase II is composed of eight subunits with molecular weights and molar ratios (in parentheses) of 170 000 (0.9), 140 000 (1.0), 43 000 (1.5), 26 000 (2.0), 22 500 (1.2), 21 500 (0.6), 18 500 (1.6) and 17 500 (2.3). The structure is closely similar to that of cauliflower RNA polymerase II.