This proposal focuses on transcription initiation and elongation by bacterial RNA polymerase (RNAP). Transcription initiation and elongation involve a series of steps: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) RNAP unwinds promoter DNA, yielding an RNAP-promoter open complex; (iii) RNAP synthesizes the first ~11 nucleotides of RNA as an RNAP-promoter initial transcribing complex, using a scrunching mechanism, in which RNAP remains stationary on promoter DNA and pulls in adjacent DNA in each nucleotide-addition cycle; and (iv) RNAP breaks its interactions with promoter DNA and synthesizes the remaining nucleotides of RNA as an RNAP-DNA elongation complex, using a stepping mechanism, in which RNAP moves forward on DNA in each nucleotide-addition cycle. Each of these steps is a potential target for transcriptional regulators Understanding transcription initiation, transcription elongation, and transcriptional regulation wil require defining the structural transitions in protein and DNA at each step, the kinetics of structural transitions, and the mechanisms by which regulators affect structural transitions. In the current period, we identified a new crystal form that enables high-resolution structural studies of the RNAP-promoter open complex (RPo) and the RNAP-promoter initial transcribing complex (RPitc), we determined the first high-resolution structure of RPo, and we delineated a DNA sequence element recognized by RNAP (the core recognition element, CRE). In other work in the current period, we developed a single-molecule-fluorescence assay that enables the monitoring of RNAP clamp conformation in solution, and we defined RNAP clamp conformation at each step in transcription initiation. The proposed work will build on the findings of the curret period. The proposed work will use x-ray crystallography, single-molecule biophysics, biochemistry, and genetics to address five specific aims: Specific Aim 1: Determination of the structural basis of de novo transcription initiation Specific Aim 2: Determination of the structura basis of initial transcription Specific Aim 3: Analysis of RNAP-CRE interactions in transcription initiation Specific Aim 4: Analysis of RNAP-CRE interactions in transcription elongation Specific Aim 5: Analysis of RNAP clamp conformation in transcription elongation The results will contribute to understanding bacterial transcription and transcriptional regulation, and will contribute to design and synthesis of small-molecule inhibitors of bacterial transcription, for use in antibacterial therapy. Since bacterial RNAP shows sequence, structural, and mechanistic similarities to eukaryotic RNAP, the results also will contribute to understanding eukaryotic transcription and transcriptional regulation.