Purification and characterization of a phosphatase from HeLa cells which dephosphorylates the C-terminal domain of RNA polymerase II.
The repetitive C-terminal domain (CTD) of RNA polymerase (RNAP) II is extensively phosphorylated concomitant with the initiation of transcription and must be dephosphorylated before RNAP II can begin another round of transcription. A CTD phosphatase was purified more than 7,500-fold from a HeLa cell extract. SDS-polyacrylamide gel electrophoresis shows a predominant protein of 205 kDa and a less abundant protein of 150 kDa co-eluting with the CTD phosphatase activity. Sedimentation and gel filtration analysis suggest that CTD phosphatase has an elongated structure with a M(r) of 200,000. This enzyme is a type 2C phosphatase in that it requires Mg2+ for activity and is resistant to okadaic acid. CTD phosphatase appears to processively dephosphorylate the CTD and is specific in that it does not dephosphorylate phosphorylase a, the alpha or beta subunits of phosphorylase kinase or RNAP II phosphorylated with casein kinase II. CTD phosphatase dephosphorylates RNAP IIO purified from calf thymus or generated in vitro by two previously described CTD kinases. These results suggest that CTD phosphatase has the properties expected for a protein phosphatase that catalyzes the conversion of RNAP IIO to RNAP IIA and may play a key role in the transcription cycle of RNAP II.
Chambers
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Section of Molecular and Cellular Biology, University of California, Davis 95616.
Dahmus
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D004988:Ethers, Cyclic / Q000494:pharmacology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008970:Molecular Weight; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000502:physiology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D012319:RNA Polymerase II / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D011189:Potassium Chloride; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase