PURIFICATION AND CHARACTERIZATION OF A PHOSPHATASE FROM HELA-CELLS WHICH DEPHOSPHORYLATES THE C-TERMINAL DOMAIN OF RNA-POLYMERASE-II

The repetitive C-termin al domain (CTD) of RNA polymerase (RNAP) II is extensively phosphorylated concomitant with the initiation of transcr iption and must be dephosphorylated before RNAP LT: 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 a nd gel filtration analysis suggest that CTD phosphatase has an elongat ed structure with a M(r) of 200,000. This enzyme is a type 2C phosphat ase 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, t he alpha or beta subunits of phosphorylase kinase or RNAP II phosphory lated with casein kinase II. CTD phosphatase dephosphorylates RNAP IIO purified from calf thymus or generated in vitro by two previously des cribed CTD kinases. These results suggest that CTD phosphatase has the properties expected for a protein phosphatase that catalyzes the conv ersion of RNAP IIO to RNAP IIA and may play a key role in the transcri ption cycle of RNAP II.