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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051853-06
Application #
6519609
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Anderson, James J
Project Start
1996-05-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2004-03-31
Support Year
6
Fiscal Year
2002
Total Cost
$146,425
Indirect Cost
Name
Indiana University Bloomington
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
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Lee, S; Carlson, T; Christian, N et al. (2000) The yeast heat shock transcription factor changes conformation in response to superoxide and temperature. Mol Biol Cell 11:1753-64
Bonner, J J; Chen, D; Storey, K et al. (2000) Structural analysis of yeast HSF by site-specific crosslinking. J Mol Biol 302:581-92
Pechatnikova, E; Taylor, E W (1999) Kinetics processivity and the direction of motion of Ncd. Biophys J 77:1003-16
Carlson, T; Christian, N; Bonner, J J (1999) A role for RNA metabolism in inducing the heat shock response. Gene Expr 7:283-91