Initiation and Elongation in T7 RNA Polymerase

点击翻译按钮获取中文摘要

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.