DNA-DEPENDENT RNA POLYMERASE-III FROM THE FUNGUS PODOSPORA-COMATA - PURIFICATION, SUBUNIT STRUCTURE AND COMPARISON WITH THE HOMOLOGOUS ENZYME OF A RELATED SPECIES

DNA-dependent RNA polymerase III was purified to homogeneity from the filamentous fungus P. comata. The enzyme was extracted at low ionic strength, separated from the polymerases I and II by DEAE-Sephadex chromatography, and purified by heparin-Sepharose and phosphocellulose chromatography; 0.1-0.2 mg highly purified homogenous enzyme with a specific activity of 220 units/mg could be obtained from 2 kg wet mycelium. The subunit composition of the enzyme was determined after sodium dodecyl sulfate polyacrylamide gel electrophoresis; 13 putative subunits of MW 174,000 (a), 129,000 (b), 87,000 (c), 50,000 (d), 39,000 (e), 23,500 (f), 21,000 (g), 19,000 (h), 17,000 (i), 16,500 (j), 13,500 (k), 11,000 (l) and 10,000 (m) were identified. All of the polypeptide components of the enzyme are present in .apprx. integral stoichiometric amounts as judged by dye binding. The presence of subunit MW = 87,000 in a molar ratio 1:1 is necessary to obtain very active enzyme. Thirteen homologous subunits were present in a preparation of RNA polymerase III from P. anserina, which is a related species. Only subunit i is different in the 2 species.