Haldenwang 9727927 The occurrence of multiple RNA polymerase specificity determinants (sigma factors) provides a cell with a powerful device for gene control. The substitution of one for another of these proteins on core RNA polymerase (RNAP) can activate or suppress a large number of genes by a single regulatory event. At least thirteen unique sigma factors are present in the bacterium Bacillus subtilis. Four of these are synthesized only during a simple differentiation (sporulation) that the bacterium undergoes in response to nutritional stress. The sporulation-specific sigma factors replace the vegetative sigmas on RNAP and are the chief activators of sporulation genes. This project seeks to determine the mechanisms by which sigma factor substitution is regulated to properly time and compartmentalize spore gene expression. Experiments are planned to examine the process by which the principal vegetative cell sigma factor (sigmaA) is excluded from RNAP and the predominant sporulation-specific sigma factor (sigmaE) is activated to replace it in just one of the two compartments that are formed by the developing cell. SigmaE is necessary for sigmaA's displacement from RNAP. In vitro experiments will measure the binding of sigmaA, sigmaE and the precursor form of sigmaE (pro-sigmaE) to RNAP to determine whether differences in the inherent affinities of each sigma for RNAP are sufficient to explain the in vivo pattern of sigma factor turnover on RNAP, or if other regulators are likely to be involved. Additional in vitro studies will ask which sigma factor activities are inhibited by the 27 amino acid "pro" sequence that is present on the precursor form of sigmaE. The pro sequence not only silences sigmaE, but also tethers pro-sigmaE to the septum that separates the sporulating cell's two compartments. Genetic and biochemical experiments will be employed to learn the elements of the "pro" sequence that are responsible for membrane binding, the proteins in the membrane with which this sequence is l ikely to interact, and whether membrane binding of pro-sigmaE is essential for its processing into sigmaE. Other genetic experiments will attempt to discover the identity of a putative forespore specific protease which appears to be responsible for limiting sigmaE accumulation in that compartment. Sporulation represents an extremely tractable model for studying sequential gene expression in a differentiating cell. The devices that Bacillus employs to communicate ongoing morphological development to its transcriptional machinery have become paradigms for how such processes could be controlled in a variety of systems. This research has at least two areas of relevance. One is in defining how cells communicate structural changes to the genes, whose activities need to be altered for the cell to properly respond to these changes. This could provide insights into how the process in which the communication could be improved, when needed for the repair of damaged tissues. A second area involves determining the basic properties of prokaryotic transcriptional regulators and the factors influencing protein stability in prokaryotic cells. These types of information may be useful in improved strategies for better expressing recombinant proteins in bacterial hosts.
