The role of RNA polymerase in gene selection during sporulation of Bacillus subtilis is being investigated by two major approaches. The first approach includes an analysis of the various RNA polymerase forms which are present in the mother cell and forespore during the sequential stages of spore formation. The enzymes will be analyzed for their subunit compositions, their interactions with various promoter-containing DNA fragments, and their specificity of transcription from various DNA templates. The second approach will include the isolation of specific DNA templates which contain promoters for RNA polymerases from the various stages of sporulation. This is being done by obtaining probes for specific sporulation genes. Antibodies are being made against sporulation-specific proteins and these antibodies will be used to detect cells synthesizing these proteins and containing plasmid cloning vehicles carrying genes for these proteins. Specific DNA fragments carrying sporulation genes will be isolated by this method and these fragments will be used as specific templates for the RNA polymerase forms found in sporulating cells. Another approach will be to use sporulation RNA polymerases as selectors of specific restricted DNA fragments. If these sporulation enzymes have a specificity for sporulation gene promoters, they should make binary complexes with these promoters preferentially. We hope to isolate specific fragments of DNA by trapping RNA polymerase-DNA complexes on nitrocellulose filters. By these studies we hope to gain an understanding of RNA polymerase structure and how it relates to selection of specific promoters.
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