Many species of bacteria posses multiple RNA polymerase sigma (sigma) factors. Association of these sigma factors with the core subunits of RNA polymerase forms holoenzymes that utilize different types of promoters. The long term goals of this proposal include understanding how sigma factors work to allow RNA polymerase to bind to specific promoters, and to determine if and how sigma factors play additional roles in the initiation of transcription subsequent to binding of the polymerase to the promoter. It is likely that the biochemical principles that govern how a sigma factor directs the bacterial RNA polymerase to use specific promoters are relevant to RNA polymerases in all organisms. Specific functions have been tentatively assigned to several regions that are highly conserved in most sigma factors. The role of these regions in sigma factors will be tested by examination of the effects of changes in the structure of sigmaE from Bacillus subtilis. SigmaE from B. subtilis is a good model for these types of studies because it is not essential for growth; therefore, alterations of its structure are not lethal.
The specific aims of this proposal include characterization of the nucleotide sequences that signal recognition of promoters by sigmaE- RNA polymerase in order to produce reagents and strains for the study of amino acid substitutions in sigmaE. This characterization will include examination of the effects of base pair substitutions in promoters, and characterization of newly-isolated promoters. The orientation and proximity of one region of sigmaE to the -10 region of promoters in polymerase-promoter complexes will be determined by examining the effects of amino acid substitutions on the use of mutant promoters, and by the use of site-specific chemical crosslinking. The effects of amino acid substitutions in sigmaE that are likely to affect steps of promoter utilization that follow binding of RNA polymerase to promoters, and those that may affect core binding will be examined in vitro. Chemical crosslinking will also be used to test the proximity of some of these residues to core subunits. Finally, the implications of a modular model of sigma structure will be tested by examining the effects of deletions and insertions in sigma factors, and examining the function of chimeric sigma factors.
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