Biochemical analysis of Pol II elongation complexes and mechanism

描述（由申请人提供）：本提案的目的是更好地了解真核生物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，负责表达真核生物中所有蛋白质编码基因的酶将在酿酒酵母模型系统中被解剖。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。对Pol II底物选择的机制以及通过控制Pol II活性的延伸因子调控Pol II的机制都没有很好的了解。该提案将确定底物特异性所需的Pol II活性位点残基，使用生化方法表征Pol II突变酶。此外，初步证据表明，延伸因子TFIIS在调节延伸过程中具有先前未被认识到的功能。TFIIS突变体允许其不同功能与Pol II分离的功能相互作用将被检查。这些实验将大大提高我们对转录过程的认识，转录过程是活细胞中基因表达的引擎。许多细胞从发育到响应环境信号的过程都有大量或必需的基因表达成分，正常的Pol II是绝对需要的。因此，在许多疾病状态下，Pol II功能被错误调节，导致异常基因表达，这可能对疾病病理至关重要。本研究特别选择研究从简单的面包酵母中提取的高度保守的Pol II。该系统为结合遗传学、生物化学和结构方法来了解Pol II功能的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在其他物种（如人类）的疾病状态中，Pol II是如何被错误调节的。

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.