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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM039917-06
Application #
3297193
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1988-04-01
Project End
1997-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Henriques, A O; Bryan, E M; Beall, B W et al. (1997) cse15, cse60, and csk22 are new members of mother-cell-specific sporulation regulons in Bacillus subtilis. J Bacteriol 179:389-98
Bryan, E M; Beall, B W; Moran Jr, C P (1996) A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis. J Bacteriol 178:4778-86
Seyler Jr, R W; Moran Jr, C P (1996) Potassium permanganate susceptibility of sigma E-RNA polymerase-promoter complexes. Gene 177:129-32
Tatti, K M; Moran Jr, C P (1996) RNA polymerase sigma factors of Bacillus subtilis: purification and characterization. Methods Enzymol 273:149-62
Tatti, K M; Moran Jr, C P (1995) sigma E changed to sigma B specificity by amino acid substitutions in its -10 binding region. J Bacteriol 177:6506-9
Tatti, K M; Shuler, M F; Moran Jr, C P (1995) Sequence-specific interactions between promoter DNA and the RNA polymerase sigma factor E. J Mol Biol 253:8-16
Diederich, B; Tatti, K M; Jones, C H et al. (1992) Genetic suppression analysis of sigma E interaction with three promoters in sporulating Bacillus subtilis. Gene 121:63-9
Jones, C H; Tatti, K M; Moran Jr, C P (1992) Effects of amino acid substitutions in the -10 binding region of sigma E from Bacillus subtilis. J Bacteriol 174:6815-21
Jones, C H; Moran Jr, C P (1992) Mutant sigma factor blocks transition between promoter binding and initiation of transcription. Proc Natl Acad Sci U S A 89:1958-62
Moran Jr, C P (1992) Transcriptional regulation of gene expression in Bacillus. Biotechnology 22:39-62

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