title: Engineered Covalent Inactivation of TFIIH-Kinase Reveals an Elongation Checkpoint and Results in Widespread mRNA Stabilization.
abstract: During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a "checkpoint" during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Juan B
authors.initials: JB
authors.lastname: Rodríguez-Molina
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Sandra C
authors.initials: SC
authors.lastname: Tseng
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Shane P
authors.initials: SP
authors.lastname: Simonett
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jack
authors.initials: J
authors.lastname: Taunton
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; The Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, USA. Electronic address: azansari@wisc.edu.
authors.forename: Aseem Z
authors.initials: AZ
authors.lastname: Ansari
mesh_terms: D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009707:Nucleosomes / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015202:Protein Engineering; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020871:RNA Stability / Q000187:drug effects; D012331:RNA, Fungal / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013997:Time Factors; D061805:Transcription Elongation, Genetic / Q000187:drug effects; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D009707:Nucleosomes; D047428:Protein Kinase Inhibitors; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases; C084210:Kin28 protein kinase, S cerevisiae; D004257:DNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase