PURIFICATION AND CHARACTERIZATION OF AN RNA-POLYMERASE-II PHOSPHATASEFROM YEAST

RNA polymerase (RNAP) II is subject to extensive phosphorylation on th e heptapeptide repeats of the C-terminal domain (CTD) of the largest s ubunit, An activity that is required for the dephosphorylation of yeas t RNAP II in vitro has been purified from a yeast whole cell extract b y >30,000-fold, The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa, The properti es of the yeast CTD phosphatase are similar to those of a previously c haracterized CTD phosphatase from HeLa cells, These properties include stimulation by the general transcription factor IIF (TFIIF), competit ive inhibition by RNAP II, magnesium dependence, and resistance to oka daic acid. Both the HeLa and yeast CTD phosphatases are highly specifi c for their cognate polymerases. Neither phosphatase functions upon th e polymerase molecule from the other species, even though the heptapep tide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are es sentially identical. The activity of the highly purified CTD phosphata se is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicatin g that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity, These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is l ikely composed of at least two components, one of which is 100 and/or 103 kDa.