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Miller, Christian (2013): Gene regulation during stress response transcription in Saccharomyces Cerevisiae: dynamic transcriptome analysis of osmotic stress response and mediator phosphorylation. Dissertation, LMU München: Fakultät für Chemie und Pharmazie
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Abstract

DYNAMIC TRANSCRIPTOME ANALYSIS MEASURES RATES OF MRNA SYNTHESIS AND DECAY IN YEAST To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.

Abstract

MEDIATOR PHOSPHORYLATION PREVENTS STRESS RESPONSE TRANSCRIPTION DURING NON STRESS CONDITIONS The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an endpoint of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast S. cerevisiae is phosphorylated at multiple sites a 17 out of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.