Transcription is the major control point of gene expression and RNA polymerase (RNAP) is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP, and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs, which have a high degree of conservation of structure and function from bacteria to man. To this end, we bring to bear a combined biochemical and biophysical approach to understand the structure and function of the RNAP in different stages of the transcription cycle, beginning with the 3.3 A-resolution X-ray crystal structure of the 380 kDa core RNAP from the thermophilic eubacterium Thermus aquaticus. This provided the basis for advances that furthered our understanding of several facets of the transcription cycle. Despite these breakthroughs, many challenges remain. Here, we propose further structural studies of transcription complexes in different stages of the transcription cycle, all aimed towards adding to our understanding of RNAP function and regulation. Specifically, we propose to: 1. Determine the crystal structure of a closed promoter complex; 2. Visualize the interaction between sigma and -10 element nontemplate DNA; 3. Determine crystal structures of binary (RNAP/DNA) and ternary (RNAP/DNA/RNA) complexes with a minimal DNA template from the M13 phage replication origin; 4. Determine the crystal structure of RNAP with the transcription elongation factor GreA/B; 5. Determine the crystal structure of T. thermophilus sigma (E) holoenzyme.
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