THE ACTIVITY OF COOH-TERMINAL DOMAIN PHOSPHATASE IS REGULATED BY A DOCKING SITE ON RNA-POLYMERASE-II AND BY THE GENERAL TRANSCRIPTION FACTORS IIF AND IIB

Each cycle of transcription appears to be associated with the reversib le phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RN AP II by CTD phosphatase, therefore, plays an important role in the tr anscription cycle. The following studies characterize the activity of HeLa cell CTD phosphatase with a special emphasis on the regulation of CTD phosphatase activity. Results presented here suggest that RNAP II contains a docking site for CTD phosphatase that is essential in the dephosphorylation reaction and is distinct from the CTD. This is suppo rted by the observations that (a) phosphorylated recombinant CTD is no t a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are competitive inhibitors of CTD phosphatase and (c) CTD phosphatase can form a stable complex with RNAP II. To test the possi bility that the general transcription factors may be involved in the r egulation of CTD phosphatase, CTD phosphatase activity was examined in the presence of recombinant or highly purified general transcription factors. TFIIF stimulates CTD phosphatase activity 5-fold. The RAP74 s ubunit of TFIIF alone contained the stimulatory activity and the minim al region sufficient for stimulation corresponds to COOH-terminal resi dues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The po tential importance of the docking site on RNAP II and the effect of TF IIF and TFIIB in regulating the dephosphorylation of RNAP II at specif ic times in the transcription cycle are discussed.