Yeast RNA polymerase C and its subunits. Specific antibodies as structural and functional probes.
Yeast RNA polymerase C purified by a simple large scale method was resolved into multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific antibodies directed against each polypeptide chain were prepared in rabbits and used as structural and functional probes. With minor exceptions, each antibody recognized specifically the corresponding polypeptide by blot-immunodetection. Cross-reactions with purified RNA polymerases A and B confirmed our previous description of the subunits shared by the three nuclear RNA polymerases. Immunoadsorption of RNA polymerase C at different stages of purification using antibodies to subunits C160 and C128 yielded the same collection of polypeptides as found in the purified enzyme: C160, C128, C82, C53, C40, C37, C34, C31, C27, C25, C23, C19, C14.5, C12.5, and C10. Subunit-specific antibodies were used to probe the activity of RNA polymerase C in a specific, reconstituted transcription system as well as on a nonspecific template. Transcription of the tRNAGlu3 gene in vitro was inhibited when RNA polymerase C was preincubated with antibodies directed to C128, C82, C53, C34, C23, or C19. Antibodies to C82, C53, and C34 were much less inhibitory in the nonspecific assay. Inhibition by anti-C128 or anti-C23 was relieved by preincubation of enzyme C with plasmid DNA prior to antibody addition. These results are discussed in terms of the participation of these polypeptides to the active enzyme molecule, and of their possible role in DNA binding or transcription factor recognition.
D000906:Antibodies; D003429:Cross Reactions; D012321:DNA-Directed RNA Polymerases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D012318:RNA Polymerase I / Q000276:immunology; D012319:RNA Polymerase II / Q000276:immunology; D012320:RNA Polymerase III / Q000032:analysis* / Q000276:immunology; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014158:Transcription, Genetic / Q000187:drug effects
D000906:Antibodies; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I; D012320:RNA Polymerase III