MOLECULAR GENETICS OF BACILLUS SUBTILIS RNA POLYMERASE

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The sporulation process of Bacillus subtilis is a model system for normal and abnormal cell development and differentiation that appears to be chiefly regulated by transcriptional control. Although the complex transcriptional apparatus of the organism is well characterized biochemically, a genetic analysis is needed to establish the physiological roles, functional interactions, and regulation of the various components. B. subtilis RNA polymerase exists in at least five cellular forms, each sharing a common, multi-subunit core enzyme and each associated with a different sigma factor that confers a characteristic promoter recognition specificity. This proposal specifically aims to characterize four genes, encoding (1) the major, 43,000 dalton sigma factor of the polymerase; (2) a minor, 37,000 dalton sigma factor that may control gene expression early in the sporulation process; (3) alpha, a core subunit; and (4) delta, a core-associated subunit that may influence transcriptional specificity. Methodology will be the same for all four genes: (1) isolate genes from a lambda expression vector bank using antibody or oligonucleotide probes; (2) confirm the identity of the gene products by independent biochemical means; (3) determine the molecular genetic organization of each cloned region by maxicell analysis, DNA sequence analysis, and S1 endonulease mapping of in vivo transcripts; (4) map the locus of the cloned gene on the B. subtilis chromosome using an integrative mapping plasmid; and (5) locate known mutations in the region on the cloned DNA, or make new mutations in the cloned genes by in vitro mutagenesis, to establish the functional domains and physiological roles of the four gene products. This research will contribute to understanding the global mechanisms regulating gene expression in B. subtilis and suggest the logic controlling developmental systems. Such understanding has a practical application in developing B. subtilis as a host for expressing and excreting cloned foreign gene products beneficial for human therapy. And on a basic level, comparing B. subtilis and E. coli gene organization and regulatory mechanisms can suggest which features of prokaryotic architecture are fundamental.