Biochemical analysis of Pol II elongation complexes and mechanism

Pol II延伸复合物的生化分析及机制

• 批准号: 7219849
• 负责人: Craig Kaplan
• 金额: $ 5.29万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7219849
• 关键词: Active Sites; Address; Appendix; Area; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Models; Cell physiology; Cells; Code; Color; Complex; Defect; Development; Disease; Elongation Factor; Enzymes; Eukaryota; Eukaryotic Cell; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Glean; Goals; Human; In Vitro; Knowledge; Life; Molecular; Nucleotides; Pathology; Phase; Polymerase; Process; Proteins; RNA; RNA Polymerase II; RNA chemical synthesis; Regulation; Resolution; Ribose; Role; Saccharomyces cerevisiae; Signal Transduction; Site; Structure; Substrate Cycling; Substrate Specificity; System; Testing; Training; Transcript; Transcription Elongation; Transcription Initiation; Transcription Process; base; in vivo; insight; mutant; pol genes; polymerization; research study; response; transcription factor; transcription factor S-II; tripolyphosphate

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.

描述（由申请人提供）：本提案的目的是更好地了解真核生物中基因表达的基本机制。具体而言，RNA聚合酶II（Pol II）的转录机制，负责真核生物中所有蛋白质编码基因的表达的酶将在模型系统酿酒酵母中进行解剖。在转录过程中，Pol II必须经历由DMA模板指导的底物选择和RNA合成的连续循环。Pol II底物选择的机制还没有很好地理解，也没有通过控制Pol II活性的延伸因子调节Pol II的机制。该提案将确定使用生化方法表征Pol II突变酶所需的底物特异性的Pol II活性位点的残基。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。将检查允许其不同功能分离的TFIIS突变体与Pol II之间的功能相互作用。这些实验将大大提高我们对转录过程的认识，这是活细胞中基因表达的引擎。从发育到对环境信号的反应的许多细胞过程具有大的或必需的基因表达组分，其中绝对需要正常的Pol II。因此，在许多疾病状态中，Pol II功能失调，导致可能对疾病病理学至关重要的异常基因表达。本研究特别选择了来自简单面包酵母S.啤酒。该系统提供了一个独特的平台，结合遗传，生物化学和结构的方法来理解Pol II功能的机制。对Pol II功能的深入了解将使我们更好地了解Pol II在其他物种（如人类）的疾病状态中可能如何被错误调节。