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.

超出所提供的空间。 了解基因调控是了解人类疾病和利用人类财富的关键。 后基因组时代出现的信息。众所周知，RNA聚合酶受到各种各样的影响 监管的阶段超越了对发起人的招募。依赖于序列的暂停、停止和终止 are knownpoints已知points点of regulation规，but are poorly知之甚少understood理解.简单的单亚基RNA聚合酶， 噬菌体T7提出了一个理想的模型系统，用于研究细胞平衡中的基本问题。 气泡形成和崩溃之间的能量学，异源双链稳定性，以及序列依赖性 易位初始二元和三元复合物的高分辨率结构的独特可用性， 这一系统提供了一个强有力的结构框架，从这个框架开始， 复合体和从初始不稳定的流产循环复合体到稳定的延伸复合体的过渡， 而功能同源性则表明， 聚合酶工程交联将启动子拴在其初始结合位点，以测试启动子是否 间隙对于向稳定和最佳功能性伸长复合物的转变是必需的。基础上 成功地理解了启动子复合物中能量上重要的相互作用， 位点特异性放置的荧光碱基类似物将映射DNA的解链和再退火， 通过观察新生异源双链体的形成和解离，在沿着 启动子清除荧光共振能量转移（FRET）和足迹将测量 启动子从其初始结合位点的置换，并测试延伸的特定结构模型 复杂.精心制作的体外选择实验将阐明序列的能量基础 转录中的依赖性停顿。衍生的延伸复合物的结构和功能表征 新出现的序列和它们的工程衍生物将直接测试单个DNA的作用 相互作用的稳定性和功能的延伸复合物。这些研究将提供基础 从而了解除了简单启动子募集之外的位点特异性转录调控。