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Saccharomyces cerevisiae

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

PROVIDER: PRJNA325832 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR3672330.fastq.gz	Fastqsanger.gz
	SRR3672340.fastq.gz	Fastqsanger.gz
	SRR3672350.fastq.gz	Fastqsanger.gz
	SRR3672360.fastq.gz	Fastqsanger.gz
	SRR3672331.fastq.gz	Fastqsanger.gz

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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

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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.

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

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

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Comparing global transcriptional changes that resulted from loss of Ypk1 or Ypk2 activity

Project description:To identify genes regulated by the AGC kinases Ypk1 and Ypk2 Experiment is designed to identify genes regulated by the kinases Ypk1 and Ypk2. These are functionally redundant AGC kinases such that yeast cells can grow in the presence of either one but the absence of both is lethal. We used a chemical genetic approach to selectively inhibit engineered ATP analog sensitive alleles (AS alleles) of each kinase. Experimental design is such that wild type (WT) Ypk1 or Ypk2 or (AS) Ypk1 or Ypk2 genes are expressed individually on plasmids in a strain background that is deleted for both Ypk1 and Ypk2 (ypk1 delta ypk2 delta). Cultures expressing these plasmids are treated with an ATP analog that is specific for AS Ypk1 or AS Ypk2. Thus, cells expressing wild type Ypk1 or Ypk2 are not sensitive but cells expressing their AS counterparts are sensitive. Arrays typically compare expression patterns in WT versus AS expressing cells at the same time point following inhibitor treatment.

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Project description:MNase-Seq for budding yeast Saccharomyces cerevisiae

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Project description:The budding yeast Saccharomyces cerevisiae is a popular host to be used to produce recombinant proteins. Here we studied three yeast strains with different productivity using the RNA-seq data to elucidate the mechanisms for improving protein production.

2020-10-26 | GSE147204 | GEO

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