Yeast DNA-dependent RNA polymerase I. A rapid procedure for the large scale purification of homogeneous enzyme

Abstract

A procedure has been developed for the rapid purification of large amounts of yeast RNA polymerase I (A). The method involves batchwise treatment with phosphocellulose and DEAE-cellulose, ion filtration chromatography on DEAE-Sephadex, sucrose gradient centrifugation, and DNA-cellulose chromatography. The enzyme obtained is apparently homogeneous by sedimentation velocity analysis and has a specific activity of 300 nmol of UMP incorporated into RNA in 10 min per mg of protein. Between 30 and 45 mg of enzyme can be obtained in 5 days from 3.0 kg of yeast cells. The subunit composition of the enzyme was determined by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. The purified polymerase is composed of 11 putative subunits with molecular weights 185,000 (Ia), 137,000 (Ib), 48,000 (Ic), 44,000 (Id), 41,000 (Ie), 36,000 (If), 28,000 (Ig), 24,000 (Ih), 20,000 (Ii), 14,500 (Ij), and 12,000 (Ik). Yeast polymerase I separates into two forms when subjected to gel electrophoresis under nondenaturing conditions. The main component which migrates faster contains all the subunits except the polypeptides Ic and If. The slow migrating component which is present in lower amounts contains all the subunits.