Initiation and Elongation in T7 RNA Polymerase

T7 RNA 聚合酶的起始和延伸

• 批准号: 7175808
• 负责人: Craig T Martin
• 金额: $ 7.78万
• 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/7175808
• 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.

超出提供的空间。 了解基因调控是了解人类疾病和开发人类财富的关键 后基因组时代产生的信息。众所周知，RNA聚合酶受多种因素的影响。 除招募发起人外的监管阶段。顺序相关的暂停、停止和终止 都是已知的监管要点，但人们对此知之甚少。一种简单的单亚基RNA聚合酶 噬菌体T7为平衡中的基本问题的研究提供了一个理想的模型系统 气泡形成和破裂、异双链稳定性和序列相关性之间的能量学 易位。初始二元和三元络合物高分辨结构的独特可用性 这个系统提供了一个强大的结构框架，可以从这个框架进入伸长研究 以及从最初的不稳定的流产循环复合体向稳定的伸长复合体的转变， 虽然功能同源性表明，所学到的基本教训将适用于所有RNA 聚合酶。工程交联会将启动子拴在它的初始结合位置上，以测试启动子 间隙是过渡到稳定和功能最佳的伸长复合体所必需的。在基础上建设 在理解启动启动子复合体中重要的能量相互作用方面取得了成功， 定点放置的荧光碱基类似物将绘制出DNA熔化和再退火的图谱，这是一致的 通过观察新生的异源双链的形成和解离，在沿路径的点 启动子许可。荧光共振能量转移(FRET)和足迹技术将测量 将启动子从其初始结合位置置换并测试延伸的特定结构模型 很复杂。精心设计的体外选择实验将阐明序列的能量基础 转录中的依赖停滞。延伸络合物的结构和功能表征 从出现的序列和它们的工程衍生品中直接测试单个DNA的作用 延长复合体的稳定性和功能中的相互作用。这些研究将提供一个基础 从中了解位点特异性转录调控，而不仅仅是简单的启动子招募。