The coordinated and timely expression of the genetic material is a crucial facet of development, growth and adaptation in all organisms. Conversely, the misappropriate expression of genes contributes to cancer and many other disease processes. In eubacteria, the precise copying of the DNA code into RNA (transcription) requires one a family of sigma factors that bind to the RNA polymerase core-subunits to allow recognition of the start points of genes (promoters). The role of sigma factors is thought to include the obligate separation of the two DNA strands prior to the transcription initiation event. The hypothesis that sigma factors act as DNA-melting proteins by providing a high affinity, single-stranded DNA binding site at the start point of transcription will be tested. This location of this binding site has been predicted from amino acid sequence comparisons between sigma factors and a family of eukaryotic single-stranded nucleic acid binding proteins (RNPs) and is thought to include aromatic amino acids that have been conserved throughout bacterial evolution. We will alter these residues genetically and determine the affects of these alterations on the biochemical functions of sigma factors. Our studies will focus on one of the major sigma factors, the Bacillus subtilis sigmaA protein, a representative alternative sigma factor, the B. subtilis sigmaD protein. The sigmaA protein will provide a biochemical system for the analysis of the affects of site- directed genetic changes on sigma factor structure and function. A complementary genetic system is available to identify altered-function alleles of sigmaD. As an additional approach to identify the location of the sigma factor: DNA interface we will make use of the observation that sigma factors become covalently crosslinked to promoter DNA when RNA polymerase:promoter complexes are exposed to ultraviolet light. The identification of the amino acids involved in forming this crosslink will help localize the regions of sigma factor in contact with DNA. These and related experiments will allow the mechanism of site-specific DNA-melting by RNA polymerase to be elucidated. It is anticipated that these studies will provide insight into the role of transcription activator proteins in facilitating the transcription initiation process and may even shed light on the analogous reactions catalyzed by eukaryotic transcription initiation proteins and by DNA replication initiation proteins.
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