Role of RNA Polymerase II Holoenzyme in Regulation

描述（申请人的摘要）：RNA聚合酶II全酶起作用。 在酵母转录调控机制中的重要作用 酿酒酵母，如其他真核生物。Srb/介体复合物， 与羧基末端结构域（CTD）相关，其功能是 将正调控输入和负调控输入整合到聚合酶。我们 最近表明全酶是高度保守的 响应葡萄糖限制的Snfl蛋白激酶信号通路。 这种新的调节相互作用提供了一个重要的新范式， 转录调控基因和生物化学分析 SnF 1与全酶的相互作用将提供对机制的深入了解。 Snfl对基因组转录模式的影响将通过DNA测序来检测。 微阵列分析，以及Snfl与适当启动子的相互作用 通过染色质免疫沉淀法测定序列。我们会研究 Snfl在影响CTD磷酸化模式变化中的作用， SrblO/Srbll激酶水平响应于葡萄糖消耗而发生。的 将鉴定与Snf 1直接相互作用全酶蛋白。MSN 3 (Std 1），一种涉及葡萄糖反应和 转录装置的特征将在于其在以下方面的作用： Snfl与全酶的物理和功能相互作用。的 Snfl和CTD激酶CTDK-I的功能关系也将被详细描述。 考察最后，我们将讨论Srb/介体复合物在 转录抑制Sfl 1，一种DNA结合蛋白， 复杂的。酵母系统为遗传分析提供了独特的优势， 在哺乳动物中保守的转录调节机制。的 建议的研究将进一步我们的功能相互作用的理解， RNA聚合酶II全酶与调节蛋白和信号 转导途径。

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.