In eukaryotes, the regulation of stress-induced genes is dependent upon the Heat Shock Transcription Factor, HSF. Recent reports show that HSF is activated by superoxide anion, O2-. O2- is produced nonenzymatically during heat shock, and by mitochondrial activity during hypoxia or recovery from anoxia. The response to O2- shows that HSF is an immediate cellular defense against reperfusion injury, incurred subsequent to ischemic stresses such as stroke. This proposal seeks to understand the mechanism of the HSF conformational change and regulation of transcriptional activation. How does HSF recognize 02-? Are specific amino acid residues modified by superoxide-and if so, which ones? How is the change in cooperativity, which involves the DNA binding domain, transmitted to the trimerization domain, and to the transcriptional activation domains to change the biological function of the protein? . These problems will be addressed through genetic manipulations and biochemical analyses. Specific mutations will be induced in the yeast HSF protein, and their effects will be determined on the superoxide-induced conformational change, and on the biological activity of HSF in vivo. Two regions within the DNA binding domain will be targeted to examine the role of these regions in the conformational change. The trimerization domain will be targeted to examine its role in the regulation of transcriptional activity, and to determine how it collaborates with the DNA binding domain to activate HSF. To move the genetic analysis onto a stronger biochemical foundation, unique cysteine residues will be put into HSF, and used to introduce probes for fluorescence resonance energy transfer. To expand the understanding of the heat shock system beyond the detailed mechanism of HSF regulation, synthetic lethal interactions will be exploited. These will identify those cellular systems that require HSF activity in the absence of stress, and thus reveal why HSF is an essential gene in yeast.
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