In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition alpha2betabeta'somega) combined with the initiation-specific sigma subunit, directs transcription initiation. Bacterial transcription depends on a primary sigma factor that is essential for viability, as well as alternative sigma's that control specific regulons. A major mechanism to control transcription initiation is through regulation of sigma activity. Dramatic insights have come from structural studies of sigma's and holoenzymes. Nevertheless many challenges remain. In this competing continuation, we propose studies to further our understanding of sigma factor structure and function, and interactions of accessory factors. Specifically, we propose to: 1. Characterize sigma factor structure and function. We will: a) Determine the structural basis for sigma interactions with the -10 element, b) Determine the structural basis for -35 element recognition by an alternative sigma, c) Probe the solution conformation of free ? using disulfide crosslinking, and d) Probe interdomain interactions of free sigma using segmental labeling and solution NMR. 2. Structurally characterize sigma/anti-sigma complexes. We will determine structures of: a) R. sphaeroides sigma/E/ChrR, and b) E. coli sigma/32/DnaK. 3. Structurally characterize interactions involved in transcription activation. We will: a) Investigate the bacteriophage lambda cl protein and the mechanism of activation, b) Investigate the role of the bacteriophage lambda cll protein in activation, and c) Determine the structure of the B. subtilis Spx/alpha-C-terminal-domain complex. 4. Structurally characterize the sigmaF regulatory system (sigmaF/SpollAA/SpollAB/SpollE) controlling the initiation of sporulation in Bacillus.
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