Role of RNA Polymerase II Holoenzyme in Regulation

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DESCRIPTION (Applicant's Abstract): The RNA polymerase II holoenzyme plays an important role in transcriptional regulatory mechanisms in the yeast Saccharomyces cerevisiae, as in other eukaryotes. The Srb/mediator complex, which is associated with the carboxy terminal domain (CTD), functions to integrate both positive and negative regulatory inputs to the polymerase. We recently showed that the holoenzyme is a direct target of the highly conserved Snfl protein kinase-signaling pathway, which responds to glucose limitation. This novel regulatory interaction provides an important new paradigm for transcriptional regulation. Genetic and biochemical analysis of the interactions of Snfl with the holoenzyme will provide insight into mechanisms. The effects of Snfl on genomic transcription patterns will be examined by DNA microarray analysis, and the interaction of Snfl with appropriate promoter sequences will be assayed by chromatin immunoprecipitation. We will examine the role of Snfl in effecting the changes in CTD phosphorylation pattern and SrblO/Srbl 1 kinase levels that occur in response to glucose depletion. The holoenzyme protein(s) that directly interact with Snfl will be identified. Msn3 (Std 1), a protein implicated in both the glucose response and the function of the transcription apparatus, will be characterized with respect to its role in the physical and functional interaction of Snfl with the holoenzyme. The functional relationship of Snfl and the CTD kinase CTDK-l will also be examined. Finally, we will address the role of the Srb/mediator complex in transcriptional repression by Sfl 1, a DNA-binding protein that interacts with the complex. The yeast system offers unique advantages for genetic analysis of transcriptional regulatory mechanisms that are conserved in mammals. The proposed studies will further our understanding of the functional interactions of RNA polymerase II holoenzymes with regulatory proteins and signal transduction pathways.