Kin28 is found within TFIIH and a Kin28-Ccl1-Tfb3 trimer complex with differential sensitivities to T-loop phosphorylation.
Basal transcription factor TFIIH phosphorylates the RNA polymerase II (RNApII) carboxy-terminal domain (CTD) within the transcription initiation complex. The catalytic kinase subunit of TFIIH is a member of the cyclin-dependent kinase (Cdk) family, designated Kin28 in Saccharomyces cerevisiae and Cdk7 in higher eukaryotes. Together with TFIIH subunits cyclin H and Mat1, Cdk7 kinase is also found in a trimer complex known as Cdk activating kinase (CAK). A yeast trimer complex has not previously been identified, although a Kin28-Ccl1 dimer called TFIIK has been isolated as a breakdown product of TFIIH. Here we show that a trimeric complex of Kin28-Ccl1-Tfb3 exists in yeast extracts. Several Kin28 point mutants that are defective in CTD phosphorylation were created. Consistent with earlier studies, these mutants have no transcriptional defect in vitro. Like other Cdks, Kin28 is activated by phosphorylation on T162 of the T loop. Kin28 T162 mutants have no growth defects alone but do demonstrate synthetic phenotypes when combined with mutant versions of the cyclin partner, Ccl1. Surprisingly, these phosphorylation site mutants appear to destabilize the association of the cyclin subunit within the context of TFIIH but not within the trimer complex.
Keogh
Michael-Christopher
MC
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Cho
Eun-Jung
EJ
Podolny
Vladimir
V
Buratowski
Stephen
S
D018844:Cyclin-Dependent Kinases; D004260:DNA Repair; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*; D013379:Substrate Specificity; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
C448995:CCL1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; C108124:TFB3 protein, S cerevisiae; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; C084210:Kin28 protein kinase, S cerevisiae