The goal of the proposed studies is to understand the fundamental mechanisms by which RNA polymerases catalyze transcription. The basic mechanisms of transcription are conserved from lower to higher organisms. The proposed studies will develop methodologies to study transcription that will aid in the understanding of more complicated bacterial and eukaryotic RNA polymerases. Through investigations of the model T7 RNA polymerase system, we hope to ultimately understand how promoter DNA sequence, RNA polymerase enzyme, and accessory factors are designed to catalyze and regulate transcription. The well-characterized T7 promoters allow a systematic study to understand promoter strength. We will continue to examine how promoter DNA sequences modulate the various steps of transcription initiation by focusing on the role of the AT-rich region of T7 promoters. The mechanism by which promoter DNA is melted by the RNA polymerase is not known partly because the initial steps of DNA binding are poorly understood. Very little is known about the conformational changes that occur in the double stranded DNA promoter and the RNA polymerase as the complex goes through various steps of initiation and promoter clearance. A major part of the proposal will focus on characterizing both the initial steps of DNA binding and structural changes in DNA and polymerase. The studies will be carried out with the following specific aims: A) To characterize the initial steps of transcription initiation and the role of DNA bending in open complex formation. B) To investigate the relationship between promoter DNA sequence and the transcriptional efficiency of the RNA polymerase. C) To characterize the conformational changes in the RNA polymerase at various stages of transcription initiation and at promoter clearance using equilibrium and stopped-flow fluorescence and luminescence resonance energy transfer methods.
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