Role of RNA Polymerase II Holoenzyme in Regulation

描述(申请人摘要)：RNA聚合酶II全酶发挥作用 酵母转录调控机制中的重要作用 酿酒酵母，和其他真核生物一样。SRB/调解人复合体， 它与羧基末端结构域(CTD)相关，其功能是 将正性和负性调节输入整合到聚合酶中。我们 最近研究表明，全酶是高度保守的直接靶标 SNFL蛋白激酶-信号通路，对葡萄糖限制作出反应。 这种新颖的监管相互作用提供了一个重要的新范式 转录调控。青枯菌的遗传和生化分析 SNFL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进行检测。 基因芯片分析，以及SNFL与适当启动子的相互作用 序列将通过染色质免疫沉淀进行分析。我们将研究 SNFL在影响CTD磷酸化模式和蛋白表达变化中的作用 在葡萄糖耗竭时发生的SRBLO/SRBL1K水平。这个 将确定与SNFL直接相互作用的全酶蛋白(S)。MSN3 (STD 1)，一种与葡萄糖反应和细胞外基质功能有关的蛋白质 转录装置，将根据其在 SNFL与全酶的物理和功能相互作用。这个 SNFL和CTD激酶CTDK-L的功能关系也将是 检查过了。最后，我们将讨论srb/调解人复合体在 与DNA相互作用的DNA结合蛋白SFL-1的转录抑制作用 这个建筑群。酵母菌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控机制。这个 提出的研究将进一步加深我们对功能相互作用的理解 RNA聚合酶II全酶及其调控蛋白和信号 转导通路。

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.