title: Mutual targeting of mediator and the TFIIH kinase Kin28.
abstract: In Saccharomyces cerevisiae, Kin28 is a member of the cyclin-dependent kinase family. Kin28 is a subunit of the basal transcription factor holo-TFIIH and its trimeric sub-complex TFIIK. Kin28 is the primary kinase that phosphorylates the RNA polymerase II (RNA pol II) C-terminal domain (CTD) within a transcription initiation complex. Mediator, a global transcriptional co-activator, dramatically enhances the phosphorylation of the CTD of RNA pol II by holo-TFIIH in vitro. Using purified proteins we have determined that the subunits of TFIIK are sufficient for Mediator to enhance Kin28 CTD kinase activity and that Mediator enhances phosphorylation of a glutathione S-transferase-CTD fusion protein, despite the absence of multiple Mediator and/or TFIIH interactions with polymerase. Mediator does not stimulate the activity of several other CTD kinases, suggesting that the specific enhancement of TFIIH kinase activity results in Kin28 being the primary CTD kinase at initiation. In addition, we have found that Kin28 phosphorylates Mediator subunit Med4 in an assay, including purified holo-TFIIH, and either Mediator or recombinant Med4 alone. Furthermore, Kin28 appears to be, at least in part, responsible for the phosphorylation of Med4 in vivo. We have identified Thr-237 as the site of phosphorylation of Med4 by Kin28 in vitro. The mutation of Thr-237 to Ala has no effect on the growth of a yeast strain under normal conditions but confirms that Thr-237 is also the site of Med4 phosphorylation in vivo.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Guidi
authors.forename: Gudrun
authors.initials: G
authors.lastname: Bjornsdottir
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Hopkins
authors.forename: Lynne
authors.initials: L
authors.lastname: Lacomis
authors.forename: Hediye
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: Paul
authors.initials: P
authors.lastname: Tempst
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Myers
mesh_terms: D047388:Casein Kinases; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins; D015966:Gene Expression Regulation, Fungal; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D051758:Transcription Factor TFIIH; D014157:Transcription Factors; D019473:Transcription Factors, TFII / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D016213:Cyclins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D011494:Protein Kinases; C093345:SSN8 protein, S cerevisiae; D047388:Casein Kinases; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases; C084210:Kin28 protein kinase, S cerevisiae; C498309:SSN3 protein, S cerevisiae; D012319:RNA Polymerase II