Biochemical analysis of Pol II elongation complexes and mechanism

描述(申请人提供)：这项建议的目的是为了更好地了解真核生物中基因表达的基本机制。具体来说，RNA聚合酶II(POL II)的转录机制将在酿酒酵母模型系统中进行剖析。POL II是负责真核生物中所有蛋白质编码基因表达的酶。在转录过程中，POL II必须经历一个由DMA模板指导的底物选择和RNA合成的连续循环。Pol II底物选择的机制还不是很清楚，控制Pol II活性的伸长因子调节Pol II的机制也不是很清楚。这一建议将使用生化方法来确定Pol II突变酶所需的底物专一性所需的Pol II活性位点残基。此外，初步证据表明，伸长因子TFIIS在调节伸长过程中具有先前未被认识的功能。将研究TFIIS突变体与POL II之间的功能相互作用，该突变体允许分离其独特的功能。这些实验将大大提高我们对转录过程的了解，转录过程是活细胞中基因表达的引擎。从发育到对环境信号的反应，许多细胞过程都有大量或必要的基因表达成分，这是绝对需要正常POL II的。因此，在许多疾病状态下，POL II功能失调，导致基因表达异常，这可能是疾病病理的关键。这项研究特别选择了从简单的面包酵母中研究高度保守的POL II。该系统为结合遗传学、生化和结构方法了解POL II的作用机制提供了一个独特的平台。对POL II功能的深入了解将有助于更好地理解POL II如何在其他物种(如人类)的疾病状态下受到错误调控。

DESCRIPTION (provided by applicant): This aims of this proposal are to better understand the basic mechanisms of gene expression in eukaryotes. Specifically, the mechanism of transcription by RNA Polymerase II (Pol II), the enzyme responsible for expression of all protein coding genes in eukaryotes will be dissected in the model system Saccharomyces cerevisiae. During transcription, Pol II must undergoes a continuous cycle of substrate selection and RNA synthesis directed by a DMA template. The mechanisms of Pol II substrate selection are not well understood nor are the mechanisms of Pol II regulation by elongation factors that control the activity of Pol II. This proposal will determine residues of the Pol II active site required for substrate specificity using biochemical approaches to characterize Pol II mutant enzymes. Additionally, preliminary evidence suggests that the elongation factor TFIIS, has previously unrecognized functions in modulating the elongation process. Functional interaction between a TFIIS mutant that allows its distinct functions to be separated and Pol II will be examined. These experiments will significantly advance our knowledge of the transcription process, which is the engine for gene expression in living cells. Many cellular processes from development to response to environmental signals have large or essential gene expression components, for which normal Pol II is absolutely required. As such, in many disease states Pol II function is misregulated causing aberrant gene expression that may be crucial for disease pathology. This study in particular chooses to study the highly conserved Pol II from the simple baker's yeast, S. cerevisiae. This system provides a unique platform for combining genetic, biochemical and structural approaches to understanding the mechanism of Pol II function. Insights into Pol II function will allow a better understanding of how Pol II may be misregulated in disease states in other species, such as humans.