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

Abstract

Each cycle of transcription appears to be associated with the reversible phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RNAP II by CTD phosphatase, therefore, plays an important role in the transcription 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 supported by the observations that (a) phosphorylated recombinant CTD is not 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 possibility that the general transcription factors may be involved in the regulation 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 subunit of TFIIF alone contained the stimulatory activity and the minimal region sufficient for stimulation corresponds to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The potential importance of the docking site on RNAP II and the effect of TFIIF and TFIIB in regulating the dephosphorylation of RNAP II at specific times in the transcription cycle are discussed.

FCP1 Phosphorylation by Casein Kinase 2 Enhances Binding to TFIIF and RNA Polymerase II Carboxyl-terminal Domain Phosphatase Activity

“…RAP74 is known to stimulate FCP1 activity (15). Indeed, addition of xRAP74 markedly increased the CTD phosphatase activity, and a half-activation was observed with 10 Ϯ 2 nM of RAP74.…”

“…First, competitive binding of general transcription factors TFIIF and TFIIB to FCP1 carboxyl-terminal domain, respectively, stimulates or inhibits its phosphatase activity (15,17). Second, free RNAP IIO molecules are better substrates than molecules engaged in transcription elongation complexes (18,19).…”

“…FCP1 is a highly specific phosphatase as the CTD within a functional RNAP II remains its only known protein substrate. FCP1 is recruited to RNAP II through docking sites on the Rpb4 subunit of RNAP II and on the RAP74 subunit of TFIIF (15,16).…”

“…CK2 Phosphorylation Enhances the Binding of FCP1 to the RAP74 Subunit of TFIIF-Genuine FCP1 is known to bind strongly to the RAP74 subunit of TFIIF, and this interaction stimulates phosphatase activity (10,15,16). To investigate a possible modulation of FCP1 binding to TFIIF by phosphorylation, a Far Western blot was performed using recombinant xRAP74 as a probe.…”

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

“…Dephosphorylation of the pol II CTD is required to regenerate the nonphosphorylated form of the enzyme for subsequent recruitment to class II nuclear gene promoters (21). The activity of this HeLa cell CTD phosphatase is stimulated by the RAP74 subunit of TFIIF, and this stimulation can be inhibited by TFIIB (22). A yeast two-hybrid screen for RAP74-interacting proteins produced partial cDNAs encoding a human CTD phosphatase, FCP1 (23,24).…”