Hyperphosphorylation of the C-terminal Repeat Domain of RNA Polymerase II Facilitates Dissociation of Its Complex with Mediator

Abstract

The Mediator complex associates with RNA polymerase II (RNAPII) at least partly via the RNAPII C-terminal repeat domain (CTD). This association greatly stimulates the CTD kinase activity of general transcription factor TFIIH, and subsequent CTD phosphorylation is involved in triggering promoter clearance. Here, highly purified proteins and a protein dissociation assay were used to investigate whether the RNAPII·Mediator complex (holo-RNAPII) can be disrupted by CTD phosphorylation, thereby severing one of the bonds that stabilize promoter-associated initiation complexes. We report that CTD phosphorylation by the serine 5-specific TFIIH complex, or its kinase module TFIIK, is indeed sufficient to dissociate holo-RNAPII. Surprisingly, phosphorylation by the CTD serine 2-specific kinase CTDK1 also results in dissociation. Moreover, the Mediator-induced stimulation of CTD phosphorylation previously reported for TFIIH is also observed with CTDK1 kinase. An unrelated CTD-binding protein, Rsp5, is capable of stimulating this CTD kinase activity as well. These data shed new light on mechanisms that drive the RNAPII transcription cycle and suggest a mechanism for the enhancement of CTD kinase activity by the Mediator complex. The Mediator complex associates with RNA polymerase II (RNAPII) at least partly via the RNAPII C-terminal repeat domain (CTD). This association greatly stimulates the CTD kinase activity of general transcription factor TFIIH, and subsequent CTD phosphorylation is involved in triggering promoter clearance. Here, highly purified proteins and a protein dissociation assay were used to investigate whether the RNAPII·Mediator complex (holo-RNAPII) can be disrupted by CTD phosphorylation, thereby severing one of the bonds that stabilize promoter-associated initiation complexes. We report that CTD phosphorylation by the serine 5-specific TFIIH complex, or its kinase module TFIIK, is indeed sufficient to dissociate holo-RNAPII. Surprisingly, phosphorylation by the CTD serine 2-specific kinase CTDK1 also results in dissociation. Moreover, the Mediator-induced stimulation of CTD phosphorylation previously reported for TFIIH is also observed with CTDK1 kinase. An unrelated CTD-binding protein, Rsp5, is capable of stimulating this CTD kinase activity as well. These data shed new light on mechanisms that drive the RNAPII transcription cycle and suggest a mechanism for the enhancement of CTD kinase activity by the Mediator complex. The C-terminal domain (CTD) 2The abbreviations used are: CTD, C-terminal repeat domain; CTDK1, CTD kinase 1; HA, hemagglutinin; E3, ubiquitin-protein isopeptide ligase; RNAPII, RNA polymerase II; TAP, tandem affinity purification. 2The abbreviations used are: CTD, C-terminal repeat domain; CTDK1, CTD kinase 1; HA, hemagglutinin; E3, ubiquitin-protein isopeptide ligase; RNAPII, RNA polymerase II; TAP, tandem affinity purification. of the largest RNA polymerase II subunit, Rpb1, is the target of dynamic phosphorylation/dephosphorylation during the transcription cycle (1Svejstrup J.Q. Biochim. Biophys. Acta. 2004; 1677: 64-73Crossref PubMed Scopus (72) Google Scholar). A non-phosphorylated version of RNAPII enters promoters (2Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1989; 264: 6693-6698Abstract Full Text PDF PubMed Google Scholar, 3Payne J.M. Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1989; 264: 19621-19629Abstract Full Text PDF PubMed Google Scholar), and the kinase activity associated with general transcription factor TFIIH somehow triggers promoter clearance by phosphorylation of serine 5 in the CTD repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) (4Feaver W.J. Gileadi O. Li Y. Kornberg R.D. Cell. 1991; 67: 1223-1230Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 5Lu H. Zawel L. Fisher L. Egly J.M. Reinberg D. Nature. 1992; 358: 641-645Crossref PubMed Scopus (330) Google Scholar, 6Trigon S. Serizawa H. Conaway J.W. Conaway R.C. Jackson S.P. Morange M. J. Biol. Chem. 1998; 273: 6769-6775Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This is thought to be followed by phosphorylation of serine 2 by the CTDK1/pTEFb kinase and serine 5 dephosphorylation by Ssu72 phosphatase during transcript elongation (7Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 8Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 9Lee J.M. Greenleaf A.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3624-3628Crossref PubMed Scopus (83) Google Scholar, 10Jones J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 11Krishnamurthy S. He X. Reyes-Reyes M. Moore C. Hampsey M. Mol. Cell. 2004; 14: 387-394Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). In all likelihood, Fcp1 phosphatase then removes the remaining CTD phosphorylation during or upon transcriptional termination, recycling the polymerase for a new round of transcription (12Archambault J. Chambers R.S. Kobor M.S. Ho Y. Cartier M. Bolotin D. Andrews B. Kane C.M. Greenblatt J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14300-14305Crossref PubMed Scopus (128) Google Scholar, 13Kobor M.S. Archambault J. Lester W. Holstege F.C. Gileadi O. Jansma D.B. Jennings E.G. Kouyoumdjian F. Davidson A.R. Young R.A. Greenblatt J. Mol. Cell. 1999; 4: 55-62Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 14Cho E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (337) Google Scholar, 15Kong S.E. Kobor M.S. Krogan N.J. Somesh B.P. Sogaard T.M. Greenblatt J.F. Svejstrup J.Q. J. Biol. Chem. 2005; 280: 4299-4306Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The Mediator complex transduces signals from sequence-specific transcriptional regulators to the general transcription machinery (16Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (251) Google Scholar, 17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar, 18Kelleher R.J. II I Flanagan P.M. Kornberg R.D. Cell. 1990; 61: 1209-1215Abstract Full Text PDF PubMed Scopus (288) Google Scholar). The association of Mediator with RNAPII, and its function in transcription, depends on the RNAPII CTD (16Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (251) Google Scholar). Mediator is able to bind an unphosphorylated glutathione S-transferase-CTD fusion protein in vitro (16Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (251) Google Scholar) and can be displaced from RNAPII using the monoclonal antibody 8WG16, which specifically recognizes the CTD repeat (17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar, 19Svejstrup J.Q. Li Y. Fellows J. Gnatt A. Bjorklund S. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6075-6078Crossref PubMed Scopus (101) Google Scholar). Apart from the CTD-interacting surfaces, Mediator also interacts with RNAPII domains outside of the CTD (20Asturias F.J. Jiang Y.W. Myers L.C. Gustafsson C.M. Kornberg R.D. Science. 1999; 283: 985-987Crossref PubMed Scopus (200) Google Scholar, 21Davis J.A. Takagi Y. Kornberg R.D. Asturias F.A. Mol. Cell. 2002; 10: 409-415Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The observation that RNAPII enters the initiation complex in the unphosphorylated (RNAPIIA) form and leaves it in the hyperphosphorylated (RNAPIIO) form gave rise to the idea that the initiation to elongation transition is somehow facilitated by hyperphosphorylation of the CTD (3Payne J.M. Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1989; 264: 19621-19629Abstract Full Text PDF PubMed Google Scholar). The association with Mediator might be one of the mechanisms by which RNAPIIA is tied to promoters. In crude yeast extracts, Mediator is almost entirely absent from the fraction containing chromatin-bound RNAPIIO engaged in transcription, whereas it is present in the soluble fraction where only RNAPIIA is detected (19Svejstrup J.Q. Li Y. Fellows J. Gnatt A. Bjorklund S. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6075-6078Crossref PubMed Scopus (101) Google Scholar). Furthermore, purified Mediator in solution is able to dynamically exchange with Mediator in immobilized holo-RNAPII (19Svejstrup J.Q. Li Y. Fellows J. Gnatt A. Bjorklund S. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6075-6078Crossref PubMed Scopus (101) Google Scholar). The idea that phosphorylation might somehow result in the dissociation of the Mediator·RNAPII complex was further supported by experiments using immobilized transcription templates in transcription-proficient yeast extracts (22Yudkovsky N. Ranish J.A. Hahn S. Nature. 2000; 408: 225-229Crossref PubMed Scopus (295) Google Scholar). In these extracts, the Mediator complex and other factors formed an activator-dependent “scaffold” for the binding of RNAPII, enabling efficient transcription initiation and re-initiation. TFIIH and were to the dissociation of RNAPII from these that phosphorylation, transcription, is for dissociation (22Yudkovsky N. Ranish J.A. Hahn S. Nature. 2000; 408: 225-229Crossref PubMed Scopus (295) Google Scholar). of the of in crude yeast that phosphorylation of RNAPII by TFIIH or dissociation of the complex (22Yudkovsky N. Ranish J.A. Hahn S. Nature. 2000; 408: 225-229Crossref PubMed Scopus (295) Google Scholar, Y. C. J. Hahn S. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). These experiments also that TFIIH protein in Mediator and that in the complex, on the of these phosphorylation be The mechanism that results in the of the Mediator complex from holo-RNAPII in a containing only RNAPII and CTD kinase. Here, the of the holo-RNAPII complex in to CTD phosphorylation using a We also the mechanism stimulation of CTD kinase activity by the Mediator complex. of yeast was using a M.S. A. II I A. A. P. 1998; 14: PubMed Scopus Google Scholar). on The with The yeast were used for protein A. Kornberg R.D. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). (17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar) and A. B. M. M. B. 1999; PubMed Scopus Google Scholar) were purified as CTD kinase was purified using a of the tandem affinity A. B. M. M. B. 1999; PubMed Scopus Google Scholar, B.P. J. Sogaard T.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). protein was by the of and The was by in and as were from the by in of and as followed by as in the were by to the was in to a of which the for was followed A. B. M. M. B. 1999; PubMed Scopus Google Scholar). Mediator was purified using a of the for A. B. M. M. B. 1999; PubMed Scopus Google Scholar). yeast was in was to the The was in 2 in protein was by the of to followed by was in to that of the for was followed A. B. M. M. B. 1999; PubMed Scopus Google Scholar). for 2 The of TFIIH, TFIIK, CTDK1, and used in this reported previously B.P. J. Sogaard T.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J.Q. W.J. J. Kornberg R.D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). holo-RNAPII and was immobilized on affinity or TFIIH, TFIIK, or CTDK1 was with or to CTD and were by and dissociation was by the in the of Mediator which was by with to Mediator and RNAPII, The holo-RNAPII from (17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar) was used as of holo-RNAPII. the dissociation assay where holo-RNAPII was immobilized via of this fraction was to in the and on a for 2 at were and the was the protein was from the by with 2 of binding 5 and the was as a as of the on to the of it by of binding containing or was and for at or at and highly purified holo-RNAPII in the was with further of of binding in dissociation this version of holo-RNAPII was then immobilized via the on this the monoclonal antibody was immobilized on to the were by and in 2 of binding to with the of holo-RNAPII from was at for with proteins were by with 2 of binding followed by of 2 of phosphorylation 5 the was in an of phosphorylation to of CTD where The were at for to and were as of of binding were with the in the was then by with of from and was by were detected by using the Mediator and were from of the monoclonal antibody which recognizes serine 2 in the CTD and with and from was to for 2 at as for binding of holo-RNAPII to were and was to in a of were by of to and at for was by of of and as was and were by The was and to a The results were and by using the CTD were in of of 5 of of RNAPII as as and was at for were by of 5 and at for were by and the was to or investigate whether CTD phosphorylation is sufficient to the association Mediator and RNAPII, used highly purified proteins in a assay The used holo-RNAPII purified from a yeast an version of one of the RNAPII was then immobilized on an affinity and a dissociation assay was This assay highly purified kinase TFIIK, or to the CTD of Rpb1, with in the of Mediator that phosphorylation of the CTD holo-RNAPII dissociation. of via CTD of RNAPII, holo-RNAPII and was to (17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar, J.Q. W.J. J. Kornberg R.D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). highly purified hyperphosphorylated RNAPII and holo-RNAPII and Surprisingly, of holo-RNAPII on antibody to protein phosphorylation The of hyperphosphorylation of the immobilized might be to as the on the C-terminal of is to the which the CTD to RNAPII P. Bushnell D.A. Kornberg R.D. Science. 2001; PubMed Scopus Google Scholar). and this the was This was in a a Mediator which previously used for Mediator M. J. Gustafsson C.M. H. Bjorklund S. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar), an from this to investigate dissociation of Mediator from immobilized RNAPII dissociation of RNAPII from immobilized In to observed previously with of holo-RNAPII complex via the on on via TFIIH to to the for hyperphosphorylated RNAPII and data the kinase is associated with holo-RNAPII S.M. J. D.A. C.M. M. Young R.A. Nature. 1995; PubMed Scopus Google Scholar). phosphorylation was on and TFIIH, that kinase activity was associated with holo-RNAPII and data these results that the be used to Mediator·RNAPII dissociation. by TFIIH of Mediator and was immobilized on antibody via the on TFIIH with or was then to dissociation. phosphorylation in the of was by the of on a that was The result of the dissociation is in dissociation of from immobilized RNAPII was by the in the of from the 2 and and in the 5 and dissociation was observed in the of of kinase the a of dissociation was observed and The and of this observation holo-RNAPII dissociation was observed in the of TFIIH of 5 and that phosphorylation of the CTD, the of TFIIH holo-RNAPII dissociation. The for observed dissociation of holo-RNAPII with the in phosphorylation of the CTD during promoter clearance (1Svejstrup J.Q. Biochim. Biophys. Acta. 2004; 1677: 64-73Crossref PubMed Scopus (72) Google Scholar). 5 is the target for phosphorylation at this with serine 2 phosphorylation only in the transcription cycle (1Svejstrup J.Q. Biochim. Biophys. Acta. 2004; 1677: 64-73Crossref PubMed Scopus (72) Google Scholar, P. E.J. Buratowski S. Genes Dev. 2000; 14: PubMed Scopus Google Scholar). CTD phosphorylation to be sufficient for dissociation in the a by other of TFIIH, as the in and also be is the of TFIIH that the CTD kinase and interacts with and in vitro W.J. Svejstrup J.Q. Kornberg R.D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). whether phosphorylation by is sufficient to holo-RNAPII the complex was purified as previously B.P. J. Sogaard T.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, observed that TFIIH phosphorylation to holo-RNAPII it was also to investigate whether dissociation phosphorylation at the specifically by this serine or whether serine 2 phosphorylation might also dissociate holo-RNAPII. We also purified the kinase that serine experiments were to the function of and CTDK1 to in the dissociation a was to of kinase was to the from immobilized on experiments that the target of and CTDK1 the activity of CTDK1 on a CTD that was with a of to serine CTDK1 a of the that the indeed in the CTD The purified were then used in the dissociation The of and is in a of Mediator is in this Mediator in the upon phosphorylation by and using highly purified TFIIK, a of Mediator was observed in the and This that the activity of the kinase complex is sufficient to dissociate holo-RNAPII in an and that the other in TFIIH an in this dissociation to in the of TFIIH and to dissociate holo-RNAPII The dissociation was this by holo-RNAPII via Mediator RNAPII and using TFIIK, CTDK1, or as the kinase the RNAPII was in this detected by the assay with and the of the polymerase by can be of kinase indeed to dissociation of RNAPII with and for and with for that phosphorylation by and CTDK1 holo-RNAPII dissociation. the CTD were and RNAPII was in the an on dissociation serine 2 and serine 5 by that immobilized holo-RNAPII can be RNAPII and Mediator by with containing In the Mediator complex its in to 2 Kim M.S. Kim Y.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In an to the of CTD phosphorylation, the dissociation assay was also using containing of in the the holo-RNAPII complex on its used with TFIIK, dissociation of Mediator was observed The was using purified CTDK1 these results that CTD phosphorylation of only serine 5 also serine 2 can result in dissociation of the Mediator·RNAPII complex. CTD and CTD-binding experiments by that Mediator stimulates the CTD kinase activity of TFIIH (17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar). The that TFIIH phosphorylation the RNAPII that Mediator might its dissociation from RNAPII during the transition from initiation to In this the of CTDK1 to dissociate the RNAPII was In to at the mechanism of CTD phosphorylation purified Mediator complex via the on and used it in RNAPII phosphorylation An the stimulation of TFIIH kinase activity is in CTD phosphorylation in to the of that TFIIH activity was the 2 to Mediator on its CTD kinase activity CTD phosphorylation by TFIIH kinase was greatly by Mediator complex as 2 and The stimulation of kinase activity by Mediator was also observed with the of TFIIH results L. Erdjument-Bromage H. Tempst P. Myers L.C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). investigate whether purified Mediator CTDK1 activity as the stimulation assay was using this kinase In these phosphorylation of RNAPII was by with the serine 2 that the was unphosphorylated in the of CTDK1 kinase. of CTDK1 in RNAPII phosphorylation and purified Mediator to the of CTDK1 kinase kinase activity and These and other data that Mediator can also CTD serine 2 phosphorylation by Rsp5, CTD-binding CTD of Mediator to the kinase activity of and CTDK1 further experiments to investigate the for the In Mediator and and CTDK1 the stimulation of kinase transcription factors the to with the We that the CTD might be for in its form with it is in with binding the that enhancement of kinase activity might also be by other CTD-binding the CTD kinase stimulation was with the interacts with the CTD J.M. J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus (180) Google Scholar), and this is for of RNAPII in to B.P. J. Sogaard T.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). The result of a CTD kinase assay using CTDK1 and is in the was as as that observed with phosphorylation of the CTD was by This result that only also an unrelated CTD-binding protein, Rsp5, can CTD of the CTD by TFIIH of Mediator from holo-RNAPII can be by the of TFIIH and previously reported that the kinase is associated with holo-RNAPII S.M. J. D.A. C.M. M. Young R.A. Nature. 1995; PubMed Scopus Google Scholar). in experiments phosphorylation dissociation of holo-RNAPII in the of of that CTD kinase activity was associated with holo-RNAPII holo-RNAPII purified from an was also with results to is to that the of Mediator with RNAPII is by CTD and J.A. Takagi Y. Kornberg R.D. Asturias F.A. Mol. Cell. 2002; 10: 409-415Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). the affinity of a Mediator module for RNAPII in a complex is by an of by the of the RNA transcript Y. H. J.A. A. Asturias F. Kornberg R.D. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), that the initiation of transcription might to of the of RNAPII from the Mediator that and bind it to promoters might be by transcript elongation and CTD phosphorylation of the TFIIH reported to with the Mediator in vitro H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). a one might that the dissociation mechanism of holo-RNAPII be on a of and CTD holo-RNAPII dissociation can be by kinase that the TFIIH for the dissociation The was by to with and W.J. Svejstrup J.Q. Kornberg R.D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). This the whether a of and CTD hyperphosphorylation was for dissociation and whether dissociation was on the of CTD that phosphorylation of the CTD by CTDK1 also holo-RNAPII. This the idea that phosphorylation is sufficient for holo-RNAPII dissociation. The that and CTDK1 can dissociate holo-RNAPII also that CTD phosphorylation phosphorylation for a Mediator is of the holo-RNAPII phosphorylation using and CTDK1 that only the RNAPII CTD is by that the dissociate holo-RNAPII by mechanisms phosphorylation of Mediator it that it as a of via and phosphorylation of the The dissociation of holo-RNAPII upon phosphorylation of serine 2 is promoter clearance associated with serine 5 hyperphosphorylation Y. M. Mol. Cell. Biol. 1996; PubMed Google Scholar). In serine 2 phosphorylation is only in the transcription cycle (1Svejstrup J.Q. Biochim. Biophys. Acta. 2004; 1677: 64-73Crossref PubMed Scopus (72) Google Scholar, 14Cho E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (337) Google Scholar). of TFIIH kinase activity a on transcription Kim J. R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, A. N. A. M. J. 2001; PubMed Scopus Google Scholar, H. S. Z. M. J. Fisher Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar), which might that CTD phosphorylation is for promoter clearance. suggest that phosphorylation to a the for TFIIH kinase activity in this This idea is supported by data that a TFIIH and CTDK1 in Y. H. J. C. S. S. Fisher Mol. Cell. Biol. PubMed Scopus Google Scholar). a of Mediator with domains can Mediator and Mediator a W. Genes Dev. 2002; PubMed Scopus Google Scholar). The hyperphosphorylated CTD Mediator in the holo-RNAPII complex to the of the observed dissociation of holo-RNAPII to be it is also that an association of Mediator with the of reported J.C. L. P. J. M. Holstege F.C. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, X. M. S. Gustafsson C.M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). This to the that Mediator somehow transcription initiation with transcriptional in the of data a for CTDK1 kinase in this or polymerase in Mediator followed by CTDK1 is the serine 2 kinase in E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (337) Google Scholar), it was reported that CTDK1 is also able to serine 5 of the yeast CTD to glutathione J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In CTDK1 specifically serine 2 RNAPII is the phosphorylation of RNAPII that was on serine 5 by a of to a in RNAPII Furthermore, used and CTDK1 to RNAPIIO S.E. Kobor M.S. Krogan N.J. Somesh B.P. Sogaard T.M. Greenblatt J.F. Svejstrup J.Q. J. Biol. Chem. 2005; 280: 4299-4306Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The serine 5-specific phosphatase Ssu72 was then used to these RNAPIIO by was whereas RNAPIIO was This that serine 5 by CTDK1 assay The of CTDK1 activity was previously reported to on J.M. W.J. Greenleaf A.L. 1997; 12: PubMed Scopus Google Scholar). in the CTDK1 the assay or the used the results and of J.C. Phatnani H.P. Haystead T.A. MacDonald J.A. Alam S.M. Greenleaf A.L. J. Biol. Chem. 2004; 279: 24957-24964Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Mediator and CTD the of the on Mediator·RNAPII the of Mediator on the activity of CTD was also in and results that the Mediator complex stimulates the CTD kinase activity of TFIIH (16Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (251) Google Scholar, 17Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar) and that it also stimulates the activity of L. Erdjument-Bromage H. Tempst P. Myers L.C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). results and these as also observed stimulation of CTDK1 kinase activity by Mediator and TFIIK, also a stimulation of CTD kinase activity by on these it is to that binding of the CTD by Mediator complex to a target to to this the CTD a that is for phosphorylation and Mediator this or somehow the CTD, phosphorylation to results the idea that kinase L. Erdjument-Bromage H. Tempst P. Myers L.C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) previously that Mediator can phosphorylation of glutathione S-transferase-CTD as as of RNAPII, that with the of the RNAPII complex for the reported Mediator and TFIIH to an in kinase as the activity of the of TFIIH the is also purified Mediator also stimulates the kinase activity of CTDK1, and CTDK1 and Mediator protein can also a CTDK1 that stimulation of CTD phosphorylation is to the Mediator complex or the kinase. stimulation of CTD phosphorylation might be a of a in CTD upon the binding of The of on CTD phosphorylation is is the for of RNAPII and the polymerase via the CTD B.P. J. Sogaard T.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, B.P. S. H. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. in Google Scholar). The of the stimulation of CTDK1 kinase activity to be We for and for on Mediator purification.