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; (iii) RNAP melts equal to approximately 14 nucleotides 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 structure of the RNAP-promoter complex at each step defining the structural transitions at each step, defining kinetics of structural transitions, and defining mechanisms by which regulators affect structural transitions. The proposed work will use fluorescence resonance energy transfer (FRET), single-molecule FRET, stopped-flow FRET, and kinetic photocrosslinking to address four specific aims:
Specific Aim 1 : To analyze the structure of RNAP holoenzyme.
Specific Aim 2 : To analyze the structures of trapped RNAP-promoter complexes.
Specific Aim 3 : To analyze the mechanism of entry of RNAP into promoter DNA.
Specific Aim 4 : To analyze the mechanism of escape of RNAP from promoter DNA. The results will contribute to understanding bacterial transcription initiation, to understanding bacterial transcriptional regulation, and to design and synthesis of low-molecular-weight inhibitors of bacterial transcription, for application in antimicrobial therapy. Since eukaryotic RNAP subunits show sequence, structural, and mechanistic similarities to bacterial RNAP subunits, the results also will contribute to understanding eukaryotic transcription initiation and regulation.
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