Prokaryotic RNA polymerases all contain a sigma subunit that directly recognizes specific sequence elements within the promoter, and by virtue of this, directs the core RNAP to the promoter. Several different sigma factors are encoded by each bacterial species to turn on transcription of different sets of genes. In E. coli, the major sigma factor sigma 7O allows transcription of most genes, while additional alternative sigma factors act to turn on a small subset of promoters, often in response to various environmental stimuli or cues. The sigma factor not only mediates the recruitment of RNAP to the promoter, it also participates actively in the initiation process, ie. in promoter melting and RNA chain initiation. The long term goal of this project is to provide a molecular understanding of how sigma factor works and how its activity is regulated. The sigma factors all contain a binding domain for the core RNAP, and they contain two specific DNA binding domains in their carboxy termini. These DNA binding domains in sigma 7O recognize hexamer elements centered respectively at -10 and -35 from the start site. In previous work, the applicant has shown that the DNA binding activity of sigma factors is intrinsically inhibited due to intramolecular interactions, involving amino terminal parts of the protein that act as specific DNA binding masks. Obviously, the DNA binding domains are unmasked when the core binds to sigma (which involves a yet distinct domain). The major goals of the proposal are (a) to understand how the two DNA binding domains communicate with each other during promoter recognition, (b) to determine how their activities are modulated by other parts of the sigma factor, and (c) to decipher how the sigma factor promotes transition of the closed promoter complex to the open complex and the ternary complex. Mutant forms of sigma factors from both the major and the alternative groups will be generated, and they will be characterized in vivo and in vitro in several respects: (i) DNA binding and interference, (ii) transcription, and (iii) specific protein-protein interactions. These studies should increase our knowledge of the basic transcription mechanisms.
