Transcription, catalyzed by RNA polymerase (RNAP), is the copying of genetic information from its DNA repository into functional RNA molecules. RNAP is a complex protein containing several subunits with distinct functions. In recent years it has become clear that the structure of RNAP has been evolutionarily conserved from bacteria to man. Our work focuses on RNAP from Bacillus subtilis, a gram positive soil microbe. Uke most bacterial RNAPs, this enzyme contains a multi-subunit core enzyme (beta beta'alpha2) associated with one of several specificity factors known as sigma. The sigma subunit determines where transcription initiates by allowing RNAP to recognize specific promoter sites and form a transcriptionally competent open (strand-separated) complex. Most transcription during growth requires the primary sigma, sigmaA. However, this sigma subunit is replaced by alternative sigma factors to allow the expression of specialized sets of genes. In addition, B. subtilis RNAP contains delta, a nonessential protein which influences enzyme activity and promoter recognition. Ultimately, we hope to understand the molecular determinants of promoter recognition, the structure of and its subunits, and transcription initiation and its regulation. The biochemical activities of sigma factor during transcription initiation will be studied by analysis of RNAP promoter complexes using biochemical and molecular genetic techniques. These experiments will address the hypothesis that a conserved region of sigma factors, region 2.3, binds to the single-stranded DNA of the transcription bubble. To test this idea, RNAP-promoter complexes will be analyzed by photocrosslinking. The role of sigma in determining the detailed pathway of transcription initiation will be investigated by comparative studies of three RNAP holoenzymes; E- sigmaA, E-sigmaD, and E-sigmaX. Finally, the role of delta in determining promoter selectivity will be studied both in vitro and in vivo. These mechanistic and structural analyses of RNAP m Bacillus subtilis will: (i) provide a necessary context for understanding transcriptional regulation in this and related organisms; (ii) provide a useful test for paradigms developed largely from study of E. coli RNAP; and (iii) allow insights into the roles of structurally related subunits of eukaryotic RNAP.
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