As structural information on the cell's macromolecular machinery accumulates, our attention turns towards understanding the dynamics of this machinery. For a multi-step reaction, such as transcription, we want to know how the macromolecular complex moves from one step to another along the reaction pathway. When alternate pathways exist (i.e., pause or not; terminate or continue; displace the RNA or form a hybrid for priming replication) we want to understand what determines the choice between these pathways and how the decision is made. Finally, we want to understand how these reaction mechanisms may be sensitive to regulation. Despite limited structural similarity between the multi-and single subunit RNAPs, the transcription reactions mediated by these enzymes are remarkably similar, even in details such as the length of the RNA at which promoter release occurs or the fine structure of terminators, suggesting that these structurally dissimilar molecules have converged upon similar solutions for executing a transcription reaction. We propose to use the structurally well characterized single-subunit 17RNAP as a model to understand the conformational dynamics of transcription. We will use nucleases and FeBABE conjugated RNAPs to probe the changes in RNAP:RNA/DNA interactions that occur as the polymerase moves from initiation to elongation. The changes in elongation complex conformation which accompany pausing, NTP binding, or formation of extended hybrids will be similarly characterized. Engineered disulphide cross-links will be used measure the effects of conformational restriction on RNAP function. The roles of sequence and supercoiling in deterrr aboutining whether the RNA is displaced, or whether extended or persistent hybrids form, will be defined. The sequence-specific interactions required for site-specific pausing will be identified. The conformational state of the active site, which is regulated via binding of T7 lysozyme, will be monitored with carboxypeptidase as the RNAP pauses. The mechanisms of promoter unwinding and RNA displacement will be characterized by mutagenizing the RNAP elements proposed to be important for these processes, and by mapping the DNA/RNA interactions made by these elements. Finally, the mechanism by which T7RNAP primes T7 DNA replication will be studied to reveal how an RNAP engaged in elongation transfers a priming transcript to a DNA polymerase.
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