title: THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing.
abstract: The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation, and, while recent studies show that inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is length dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.
authors.affiliation: Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Nilson
authors.affiliation: Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Jiannan
authors.initials: J
authors.lastname: Guo
authors.affiliation: Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Turek
authors.affiliation: Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Brogie
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Delaney
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Donal S
authors.initials: DS
authors.lastname: Luse
authors.affiliation: Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA; Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA. Electronic address: david-price@uiowa.edu.
authors.forename: David H
authors.initials: DH
authors.lastname: Price
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D010655:Phenylenediamines / Q000737:chemistry* / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000737:chemistry* / Q000494:pharmacology; D012319:RNA Polymerase II / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D061785:Transcription Initiation, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000378:metabolism; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D000970:Antineoplastic Agents; C411985:NSMF protein, human; D009687:Nuclear Proteins; D010655:Phenylenediamines; D011743:Pyrimidines; C112224:SUPT5H protein, human; C000596184:THZ1 compound; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human