Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties.
DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.
D000818:Animals; D000936:Antigen-Antibody Complex; D002460:Cell Line; D004256:DNA Polymerase I / Q000302:isolation & purification; D004257:DNA Polymerase II / Q000302:isolation & purification; D004258:DNA Polymerase III / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D007106:Immune Sera; D005779:Immunodiffusion; D051379:Mice; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology*; D010954:Plasmacytoma / Q000201:enzymology; D014158:Transcription, Genetic; D014981:Xenopus
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D012321:DNA-Directed RNA Polymerases; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III