Investigator's application) The regulatory response of cells to temperature changes is highly conserved among all organisms. However, little is known about the signal transduction pathway which allows cells to respond to temperature changes or the precise functions of the heat shock proteins (hsps) induced by heat. The long term goal is to understand the nature of the cellular response to heat by investigating this system in Escherichia coli. In E. coli sigma 32 transcribes the heat shock genes. The amount and activity of sigma 32 is altered by temperature changes. A regulatory loop sensing changes in temperature includes the DnaK, DnaJ and GrpE (K,J,E) hsps. A major focus of the research during the current grant period will be to dissect the regulatory network by looking for additional gene products that are involved in the signal transduction pathway and by studying how the K, J, and E heat shock proteins regulate sigma 32. One outcome of these studies will be a structure function analysis of K,J, and E. Because both K and J have highly conserved eukaryotic analogues, this information is likely to be relevant to both the function and regulation of heat shock proteins in higher organisms. Recently, Dr. Gross has found that another sigma factor, sigma E, also transcribes genes required at high temperature. The final goal will be to determine how sigma E is regulated by temperature upshift and to understand the function of sigma transcribed genes in high temperature physiology. Thus, the specific aims are: 1. to define the signal responsible for increasing the amount of sigma 32 after temperature increase. Strains that are unable to signal or that signal continuously will be selected. The proposition that a part (or all) of the signal is a drop in the pool of free K, J, and E will be explored. 2. To define the functional domains of K,J, and E, a mutational and biochemical analysis will be used. The intimate connection between K, J, and E function and heat shock regulation allows the selection of dominant negative mutations. A combination of in vivo and in vitro characterization will permit dissection of functional organization of the K,J, and E proteins. Because K and J have highly conserved eukaryotic analogues, this information is likely to be relevant to the function of hsps in eukaryotes. 3. To understand how sigma 32 degradation is regulated, the protease(s) degrading sigma 32 will be identified and their biological role examined. An in vitro assay for sigma 32 degradation will enable investigation of how K,J, and E modulate proteolysis. 4. To understand how the activity of sigma 32 is reduced after temperature downshift, sigma 32 determinants allowing inactivation will be identified, and an in vitro system to define the components and mechanism of the reaction will be set up. 5. To define the role of sigma E in high temperature physiology, sigma E will be cloned, it will be determined whether the amount or the activity of sigma E is regulated by temperature upshift and the function of the sigma E regulon will be explored.
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