Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II.
Mammalian cells contain two subspecies of RNA polymerase II, designated IIO and IIA. The objectives of these studies were to determine the structural relationship between these subspecies and to determine the functional significance of these differences. Subunits IIo and IIa were purified from calf thymus, and the effect of alkaline phosphatase treatment on electrophoretic mobility and immunochemical reactivity was examined. The removal of phosphate converts subunit IIo to a form indistinguishable from that of subunit IIa. These results indicate that subunit IIo is produced by multisite phosphorylation of subunit IIa. The distribution of phosphate within subunit IIo was determined by CNBr cleavage of in vivo labeled HeLa cell RNA polymerase II. 32P-Labeled subunit IIo was purified by immunoprecipitation and cleaved with CNBr, and the resultant peptides were analyzed. The quantitative recovery of 32P in the C-terminal peptide establishes that this domain is the primary site of phosphorylation. In an effort to assess the level of phosphorylation of the transcriptionally active form of RNA polymerase II in HeLa nuclei, transcription was carried out in the presence of 4-thiouracil triphosphate and the nascent labeled transcript cross-linked to RNA polymerase. Specific photoaffinity labeling of subunit IIo was observed. Alkaline phosphatase treatment results in an increase in the mobility of photoaffinity labeled subunit IIo to approach that of subunit IIa. These results indicate that subunit IIo is a component of transcriptionally active RNA polymerase II.
D000469:Alkaline Phosphatase; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D013950:Thymus Gland / Q000201:enzymology; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011494:Protein Kinases; D012319:RNA Polymerase II; D000469:Alkaline Phosphatase