Wheat leaf RNA polymerases: III. Purification of the soluble RNA polymerase

Abstract

A procedure has been developed for the purification of a soluble DNA-dependent RNA polymerase from wheat seedling leaves. The RNA polymerase activity present in the high-speed supernatant fraction from the initial tissue homogenate was purified by ammonium sulfate precipitation and chromatography on diethylaminoethyl cellulose, phosphocellulose, and Bio-Gel A15m. The purified enzyme shows one polypeptide component with a molecular weight of 65,000 on electrophoresis in 0.1% sodium dodecylsulfate-5% polyacrylamide gels but exists in a highly aggregated form ( M r 15×10 6 or greater) at low ionic strength. At high Tris concentrations the RNA polymerase can be reversibly converted to a lower molecular-weight form. The purified RNA polymerase is dependent on added DNA and Mg 2+ for activity. Maximum activity requires ATP, GTP, CTP, and UTP and the product is alkali labile and digested by pancreatic RNAase. The purified RNA polymerase is inhibited by pyrophosphate, but not by phosphate, and is inhibited by rifamycin-SV, but only at very high concentrations. Other protein-specific inhibitors of RNA polymerase, namely, α -amanitin, streptovaricin, and streptolydigin, do not inhibit the enzyme at high concentrations. The wheat leaf soluble RNA polymerase resembles the Neurospora crassa and rat liver mitochondrial RNA polymerases in subunit composition, subunit molecular weight, and in the formation of aggregates of high molecular weight.