Initiation and Elongation in T7 RNA Polymerase

T7 RNA 聚合酶的起始和延伸

• 批准号: 6830191
• 负责人: Craig T Martin
• 金额: $ 23.09万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6830191
• 关键词: DNA directed RNA polymerase; DNA footprinting; RNA biosynthesis; active sites; bacteriophage T7; chemical kinetics; deoxyribonucleoside triphosphate; enzyme activity; enzyme mechanism; enzyme structure; genetic promoter element; genetic regulation; nucleoproteins; phosphoric ester; protein structure function; site directed mutagenesis; stop flow technique; temperature; thermodynamics; transcription factor

EXCEED THE SPACE PROVIDED. Understanding genetic regulation is key to understanding human disease and to exploiting the wealthof information arising in the post-genomic era. It is well known that RNA polymerases are subject tovarious stages of regulation beyond recruitment to the promoter. Sequence dependent pausing, arrest, and termination are knownpoints of regulation, but are poorly understood. The simple single subunitRNA polymerase from bacteriophage T7 presents an model ideal system for the study of fundamental issues in the balance of energetics between bubble formation and collapse, heteroduplex stability, and sequence dependent translocation. The unique availability of high resolution structures of initial binary and ternary complexes in this system provides a powerful structural framework from which to move into studies of the elongation complex and the transition from an initial unstable abortive cycling complex to a stable elongation complex, while functional homologies suggest that the underlyinglessons learned will be applicable to all RNA polymerases. Engineered crosslinks will tether the promoter to its initial binding site to test whetherpromoter clearance is necessary for the transition to a stable and optimally functional elongation complex. Building on successes in understandingenergetically important interactions in the initiatingpromoter complex, site-specifically placed fluorescent base analogs will map melting and reannealing of the DNA,coincident with observation of formation and dissociation of the nascent heteroduplex, at points along the path of promoter clearance. Fluorescence resonance energy transfer (FRET) and footprinting will measure displacement of the promoter from its initial binding site and test specific structural models of theelongation complex. Carefully crafted in vitro selection experiments will elucidate the energetic basis of sequence dependent stalling in transcription. Characterization of structure and function in elongation complexes derived from emergent sequences and from their engineered derivatives will directly test the roles of individual DNA interactions in the stability and function of the elongation complex. These studies will provide a foundation from which to understand site specific transcriptional regulation beyond simple promoter recruitment. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。了解基因调控是了解人类疾病和利用后基因组时代产生的大量信息的关键。众所周知，RNA 聚合酶除了募集到启动子之外还要经历各个阶段的调节。序列依赖性暂停、停止和终止是已知的调节点，但人们对其了解甚少。来自噬菌体 T7 的简单单亚基 RNA 聚合酶为研究气泡形成和破裂之间的能量平衡、异源双链体稳定性和序列依赖性易位之间的基本问题提供了一个理想的模型系统。该系统中初始二元和三元复合物的高分辨率结构的独特可用性提供了强大的结构框架，从该框架开始研究延伸复合物以及从初始不稳定的中止循环复合物到稳定的延伸复合物的转变，而功能同源性表明所学到的基本经验将适用于所有RNA聚合酶。工程交联将启动子束缚在其初始结合位点，以测试启动子清除对于转变为稳定且最佳功能的延伸复合物是否是必要的。基于成功理解起始启动子复合物中能量重要的相互作用，位点特异性放置的荧光碱基类似物将绘制 DNA 的熔化和重新退火图，与观察启动子清除路径沿线点处的新生异源双链体的形成和解离相一致。荧光共振能量转移（FRET）和足迹法将测量启动子从其初始结合位点的位移并测试延伸复合物的特定结构模型。精心设计的体外选择实验将阐明转录中序列依赖性停滞的能量基础。对源自出现序列及其工程衍生物的延伸复合物的结构和功能的表征将直接测试个体DNA相互作用在延伸复合物的稳定性和功能中的作用。这些研究将为了解简单的启动子招募之外的位点特异性转录调控奠定基础。表演网站==========================================章节结束===============================================