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Pol II and CTD Phosphorylation ChIP-chip of ATP-analog sensitive Kin28 budding yeast

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.

ORGANISM(S): Saccharomyces cerevisiae BY4741 Saccharomyces cerevisiae

PROVIDER: GSE83412 | GEO | 2016/06/16

SECONDARY ACCESSION(S): PRJNA325831

REPOSITORIES: GEO

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Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast

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Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast

Project description: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 â??checkpointâ?? 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. Total RNA was collected from wild-type and analog-sensitive Kin28 strains treated with reversible inhibitor 1-NAPP-1, irreversible inhibitor CMK, and solvent control DMSO. Equivalent ratios of S. pombe : S. cerevisiae cells were added to each sample before RNA extraction for normalization of read counts after sequencing. Nascent RNA was purified from total RNA by 4-thiouracil labeling, biotinylation, and streptavidin-pulldown. As a negative control, nascent RNA was also extracted from total RNA from cells that had not been treated with 4-thiouracil.

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Pol II and CTD Phosphorylation of ATP-analog sensitive Kin28 budding yeast

Project description:Pol II and CTD Phosphorylation of ATP-analog sensitive Kin28 budding yeast

| PRJNA325829 | ENA

Dynamic turnover of paused pol II complexes at human promoters

Project description:Paused RNA polymerase II that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused pol II is a stable or a dynamic target remains unresolved. We report that most 5’-paused pol II throughout the genome is turned over within 2 minutes. This process is revealed under hypertonic conditions that prevent pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel pre-initiated state of pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that pol II occupancy near 5’ ends is governed by a cycle of ongoing assembly of pre-initiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause, is by escape from premature termination.

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