The spatiotemporally controlled exchange of different protein complexes that act on RNA polymerase II (Pol II), chromatin and the nascent transcripts during the transcription cycle is orchestrated via post-translational modifications of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of Pol II. One model of how the CTD might mediate its functions is inspired by the so- called histone code. The CTD code hypothesis potsi that different patterns of post-translational modifications on the CTD encipher a code that is read by different protein complexes involved in Pol II dependent RNA biogenesis. How the CTD orchestrates the dynamic association of relevant protein complexes at different classes of genes is a fundamentally important question that lies at the intersection of transcriptional regulation, co-transcriptional processing, establishment of chromatin states, genome stability, and etiology of many human diseases. Of the enzymes that encipher the CTD code, Bur1/Cdk9, a cyclin dependent kinase, isparticularly interesting. It was discovered as a positive transcription elongation factor -b (P-TEFb) that was essential for the release of an early elongation block placed on HIV-1 transcription. Recently, importance of CTD patterning and the physiological role of Bur1/Cdk9 in meaningfully patterning of the CTD has been debated. To address this controversy, we have devised a suite of new approaches to comprehensively examine Bur1/Cdk9 specificity and activity in vitro and in vivo. We employ a highly specific chemical targeting approach to irreversibly inhibit Bur1 in order to resolve the highly debated issue of the importance of Bur1 in placing gene-class specific CTD marks in vivo, we then apply a sophisticated ETD-based phospho-proteomic approach and our newly developed high density peptide/substrate microarrays to comprehensively define the role of CTD priming by prior marks in guiding Bur1/Cdk9 in the subsequent placement of specific phospho-CTD patterns. Then, we describe a strategy that leverages our uniquely tagged CTD strains to determine whether Bur1 places different marks on different CTD repeats in the context of actively transcribing Pol II in vivo, and via robotically automated high throughput methods, we propose to elucidate whether any member of the set of ~90 known Bur1 binding partners alters the specificity or activity of the kinase in vitro and CTD patterning in a gene-class specific manner i vivo. Taken together we outline an innovative proposal that seamlessly integrates a chemical, biochemical, genetic, genomic, proteomic and computational suite of tools to elucidate the molecular cues that guide CTD patterning by Bur1/Cdk9, an essential kinase that is conserved across eukaryotes and is a highly validated therapeutic target for several devastating human ailments.

Public Health Relevance

Regulated expression of eukaryotic genes requires a highly orchestrated series of events that converge on the transcription of genomic information into RNA by DNA directed RNA polymerase II. The c-terminal domain of the largest subunit of this polymerase is modified in specific patterns by several enzymes, one of which is essential for cellular survival. We describe a multi-disciplinary strategy that relies on chemical, biological, genomic and computational approaches to decipher the cues that guide the specific patterning and function of the polymerase.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics A Study Section (MGA)
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Sledjeski, Darren D
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University of Wisconsin Madison
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Nemec, Corey M; Singh, Amit K; Ali, Asfa et al. (2018) Noncanonical CTD kinases regulate RNA polymerase II in a gene-class-specific manner. Nat Chem Biol :
Erwin, Graham S; Grieshop, Matthew P; Ali, Asfa et al. (2017) Synthetic transcription elongation factors license transcription across repressive chromatin. Science 358:1617-1622
Nemec, Corey M; Yang, Fan; Gilmore, Joshua M et al. (2017) Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner. Proc Natl Acad Sci U S A 114:E3944-E3953
Rodríguez-Molina, Juan B; Tseng, Sandra C; Simonett, Shane P et al. (2016) Engineered Covalent Inactivation of TFIIH-Kinase Reveals an Elongation Checkpoint and Results in Widespread mRNA Stabilization. Mol Cell 63:433-44