Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.
Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D046911:Macromolecular Substances; D010085:Oxidative Phosphorylation; D010710:Phosphates / Q000378:metabolism; D011494:Protein Kinases / Q000302:isolation & purification; D012318:RNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D012320:RNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D010710:Phosphates; D011494:Protein Kinases; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I; D012320:RNA Polymerase III