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| Status |
Public on Jun 16, 2016 |
| Title |
Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
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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| Contributor(s) |
Ansari AZ, Tseng SC |
| Citation(s) |
27477907 |
| |
| Submission date |
Jun 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Aseem Ansari |
| Organization name |
University of Wisconsin-Madison
|
| Department |
Biochemistry and The Genome Center
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| Lab |
Ansari
|
| Street address |
440 Henry Mall
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL13821 |
Illumina HiSeq 2000 (Saccharomyces cerevisiae) |
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| Samples (34)
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| This SubSeries is part of SuperSeries: |
| GSE83417 |
Pol II and CTD Phosphorylation of ATP-analog sensitive Kin28 budding yeast |
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| Relations |
| BioProject |
PRJNA325832 |
| SRA |
SRP076628 |
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