Purification and subunit analysis of wheat-germ ribonucleic acid polymerase II

Abstract

A procedure is described for the purification of the alpha-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH(4))(2)SO(4), chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradient centrifugation. Under denaturing conditions the enzyme dissociated into five polypeptides with molecular weights and molar ratios of 220000 (0.9), 170000 (0.1), 140000 (1.0), 45000 (0.2), and 40000 (0.4). Approx. 1mg of purified RNA polymerase was obtained as a routine from 100g of starting material.