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.

2016-06-16 | E-GEOD-83416 | biostudies-arrayexpress

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

2016-06-16 | GSE83416 | GEO

Budding yeast RNA polymerase II CTD phosphorylation and transcription termination factor localization

Project description:Whole genome analysis of total RNA pol II, Ser2-, Ser5- and Ser7-phosphorylated RNA pol II, in WT and mutants of the C-terminal domain (CTD) kinases Ctk1 and Kin28, and localization of the termination factors Pcf11, Nrd1 and Rat1. ChIP-chip using ligation-mediated PCR-amplified material hybridized to NimbleGen 385K arrays (50mers, median probe spacing 32 bp, cat. No. C4214-00-01).

2010-09-15 | E-GEOD-23960 | biostudies-arrayexpress

Transient transcriptome sequencing (TT-Seq) and 5'P-Seq of ATP-analog sensitive Kin28 budding yeast

Project description:Transient transcriptome sequencing (TT-Seq) and 5'P-Seq of ATP-analog sensitive Kin28 budding yeast

| PRJNA516282 | ENA

Budding yeast RNA polymerase II CTD phosphorylation and transcription termination factor localization

2010-09-15 | GSE23960 | GEO

Transient transcriptome sequencing (TT-Seq) and 5'P-Seq of ATP-analog sensitive Kin28 budding yeast

Project description:Inhibition of Kin28/CDK7, the kinase subunit of TFIIH, leads to defects in transcription of protein-coding genes. Despite a severe reduction in nascent RNA synthesis, the majority of mRNAs retain their steady-state level upon inhibition. In this study, we examined the determinants of mRNA stability in cells experiencing transcriptional crisis via irreversible chemical inhibition of Kin28. We discovered that the inhibited Kin28 transcriptome resembles the transcriptome of cells treated with an inhibitor of protein synthesis. Indeed, inhibition of Kin28 induces a coordinated decrease in translation and an increase in P-body formation. Unexpectedly, integrated stress response effectors do not trigger the observed proteostasis and ribostasis. Rather, mRNAs that are buffered from degradation display a preference for Ski2 over Nab2 and are differentially sensitive to the 5′-exonuclease Xrn1. These findings reveal that a nuclear kinase, well-known for its role in early stages of RNA synthesis, orchestrates multiple molecular processes in different cellular compartments.

2022-01-18 | GSE125409 | GEO

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

Project description:This SuperSeries is composed of the SubSeries listed below.

2016-06-16 | GSE83417 | GEO

Transcriptional profiling of Neurospora crassa cells containing MAK2Q100G allele: Exposure to ATP-analog (1NM-PP1) vs control

Project description:To study the consequences of MAK-2 activity modulation during vegetative cell fusion, we took advantage of a previously constructed allele of MAK-2 (MAK-2Q100G) to specifically perturb kinase signaling during germling vegetative cell fusion (inhibition of MAK-2Q100G activity by addition of the ATP analog 1NM-PP1 results in a phenotype indistinguishable from mak-2 deletion strains). Whole genome microarrays of mak-2Q100G cells following 20 min 1NM-PP1 treatment were performed.

2013-06-01 | GSE46912 | GEO

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