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 has been purified to homogeneity from the filamentous fungus Podospora 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 homogeneous 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 sulphate/polyacrylamide gel electrophoresis; thirteen putative subunits of molecular weight 174000 (a), 129000 b), 87000 (c), 50000 (d), 39000 (e), 23500 (f), 21000 (g), 19000 (h), 17000 (i), 16500 (j), 13500 (k), 11000 (l) and 10000 (m) were identified. All of the polypeptide components of the enzyme are present in about integral stoichiometric amounts as judged by dye binding. The presence of subunit Mr = 87000 in a molar ratio 1:1 is necessary to obtain very active enzyme. Thirteen homologous subunits were observed in a preparation of RNA polymerase III from Podospora anserina, which is a related species. Only subunit i is different in the two species.
D001203:Ascomycota / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D012320:RNA Polymerase III / Q000302:isolation & purification*; D013045:Species Specificity; D013098:Xylariales / Q000201:enzymology*
D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III