Transcription initiation by bacterial RNA polymerase (RNAP) involves a series of steps: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) RNAP clamps tightly onto promoter DNA, yielding an RNAP-promoter intermediate complex; (in) RNAP unwinds -14 base pairs of promoter DNA, yielding a catalytically competent RNAP-promoter open complex; (iv) RNAP initiates synthesis of RNA, yielding an RNAP-promoter initial transcribing complex; and (v) RNAP breaks its interactions with promoter DNA-""""""""escapes""""""""-yielding an RNAP-DNA elongation complex. Each of these steps is a potential target for transcriptional regulators. Understanding transcription initiation and transcriptional regulation will require defining the structural transitions in protein and DNA at each step, defining kinetics of structural transitions, and defining mechanisms by which transcriptional regulators affect structural transitions. ? ? The proposed work will use kinetic photocrosslinking, single-molecule fluorescence resonance energy transfer, single-molecule nanomanipulation, and chromatin immunoprecipitation to address four specific aims: ? Specific Aim 1: To analyze formation of protein-DNA interactions during promoter entry. ? Specific Aim 2: To analyze breakage of protein-DNA interactions during promoter escape. ? Specific Aim 3: To analyze DNA structural transitions during promoter entry and escape. ? Specific Aim 4: To analyze RNAP structural transitions during promoter entry and escape. ? ? In addition, the proposed work will use integrated crystallographic, biophysical, biochemical, genetic, and combinatorial-chemistry approaches to address two specific aims: ? ? Specific Aim 5: To characterize known small-molecule inhibitors of RNAP. ? Specific Aim 6: To identify and characterize novel small-molecule inhibitors of RNAP. ? ? The results will contribute to understanding bacterial transcription initiation and regulation, will provide tools for analysis of bacterial transcription initiation and regulation, and will provide lead compounds for development of antibacterial therapeutic agents. Since eukaryotic RNAP shows sequence, structural, and functional similarities to bacterial RNAP, the results also will contribute to understanding eukaryotic transcription initiation and regulation. ? ?
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