T7RNAP is the most well studied member of a widespread class of RNAPs that includes phage-encoded RNAPs, plastid and plasmid encoded enzymes, and chloroplast and mitochondrial RNAPs. It also displays more limited sequence similarity, but extensive structural similarity, to DNAPs, RTs & RNA-directed RNAPs. T7RNAP plays multiple roles in the T7 life cycle. In addition to transcribing the phage genes, it primes T7 DNA replication and probably acts to initiate assembly of the T7DNA packing and maturation machinery at the T7 concatemer junction. The relatively simple single-subunit T7RNAP appears able to carry out these complex reactions--usually executed by much larger multi-subunit enzymes--because it of its extraordinary conformational adaptability. Because of its relative structural simplicity, mechanistic complexity, and the advanced state of our understanding of this molecule, T7RNAP presents an exceptionally attractive system in which to pursue a central ambition of molecular biology: understanding the mechanisms of the machinery that carries out cellular processes. Over the next project period we will address the outstanding questions which have yet to be tackled, or have received conflicting answers, in this system. Specifically, we will characterize the structural transitions undergone by the transcription complex during the late stages of initial transcription and the transition from the immature to the mature EC. We will ask how the force that ruptures the promoter:RNAP interaction at the point of promoter release is generated, and characterize the role of individual amino acids in translocation during elongation. Conformational changes during pausing and termination at the T7 concatemer junction and the T7 Tphi (hairpin) terminator will be studied with approaches that allow time-resolved characterization of transient complexes. Finally, T7RNAP priming of T7 DNAP will be characterized with the goal of answering the following questions: how is the primer transferred from RNAP to DNAP and how does the sequence of the primary origin contribute to this process?
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