Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis.
RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D011494:Protein Kinases; C026813:protein kinase NII; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases