Initiation and Elongation in T7 RNA Polymerase

超出提供的空间。了解基因调控是理解人类疾病和利用后基因组时代产生的丰富信息的关键。众所周知，RNA聚合酶受到不同阶段的调控，而不是招募到启动子。顺序依赖的停顿、停顿和终止是已知的调节点，但人们对此知之甚少。来自T7噬菌体的简单的单亚单位RNA聚合酶为研究泡泡形成和破裂、异源双链稳定性和序列依赖易位之间的能量学平衡的基本问题提供了一个理想的模型系统。在该体系中，初始二元和三元配合物的高分辨结构的独特可用性为研究延伸复合体提供了一个强大的结构框架，以及从最初不稳定的流产环复合体到稳定的延伸复合体的转变，而功能同源性表明，所学到的基本经验教训将适用于所有RNA聚合酶。工程交联会将启动子连接到其初始结合位置，以测试启动子清除是否是向稳定和功能最佳的延长复合体过渡所必需的。在成功理解启动启动子复合体中重要的能量相互作用的基础上，定点放置的荧光碱基类似物将绘制DNA的熔融和再退火图，这与观察到新生的异源双链的形成和解离一致，在启动子清除的路径上。荧光共振能量转移(FRET)和足迹技术将测量启动子从其初始结合位置的位移，并测试延伸复合体的特定结构模型。精心设计的体外选择实验将阐明转录中序列依赖停滞的能量基础。从新出现的序列及其工程衍生物衍生的延伸复合体的结构和功能的表征将直接测试单个DNA相互作用在延伸复合体的稳定性和功能中的作用。这些研究将为理解简单的启动子招募以外的位点特异性转录调控提供基础。表演网站========================================Section End===========================================

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===========================================