Pol II and CTD Phosphorylation of ATP-analog sensitive Kin28 budding yeast
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae BY4741 Saccharomyces cerevisiae
PROVIDER: GSE83417 | GEO | 2016/06/16
SECONDARY ACCESSION(S): PRJNA325829
REPOSITORIES: GEO
Similar Datasets
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 “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.
2016-06-16 | GSE83412 | GEO
Project description:Pol II and CTD Phosphorylation of ATP-analog sensitive Kin28 budding yeast
| PRJNA325829 | ENA
Project description:Pol II and CTD Phosphorylation ChIP-chip of ATP-analog sensitive Kin28 budding yeast
| PRJNA325831 | ENA
Project description:Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is cotranscriptionally recruited to the 5' end of ARG1 in a manner stimulated by interaction of the BUR1 C terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5 and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.
| S-EPMC2683426 | biostudies-literature
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
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 “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.
2016-06-16 | GSE83416 | GEO
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
Project description:Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.
| S-EPMC3419927 | biostudies-literature
Project description:Rho GTPases, including the Rho, Cdc42, Rac, and ROP subfamilies, act as pivotal signaling switches in various growth and developmental processes. Compared with the well-defined role of cytoskeletal organization in Rho signaling, much less is known regarding transcriptional regulation. In a mutant screen for phenotypic enhancers of transgenic Arabidopsis plants expressing a constitutively active form of ROP2 (designated CA1-1), we identified RNA polymerase II (Pol II) C-terminal domain (CTD) phosphatase-like 1 (CPL1) as a transcriptional regulator of ROP2 signaling. We show that ROP2 activation inhibits CPL1 activity by promoting its degradation, leading to an increase in CTD Ser5 and Ser2 phosphorylation. We also observed similar modulation of CTD phosphorylation by yeast Cdc42 GTPase and enhanced degradation of the yeast CTD phosphatase Fcp1 by activated ROP2 signaling. Taken together, our results suggest that modulation of the Pol II CTD code by Rho GTPase signaling represents an evolutionarily conserved mechanism in both unicellular and multicellular eukaryotes.
| S-EPMC5167197 | biostudies-literature
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.
2010-09-15 | GSE23960 | GEO