The Heat Shock Transcription Factor, HSF, regulates transcription of genes encoding the protein chaperones known as heat shock proteins (hsps). HSF stimulates hsp expression at a low level in unstressed cells, and at a high level in cells subjected to hyperthermic stress. In the yeast, Saccharomyces cerevisiae, HSF activity depends on the conformation of HSF_at low and high temperatures, HSF adopts conformations that are distinctly different. In the low-temperature conformation, its transcriptional activation domains are masked; in the high-temperature conformation, they are exposed. To date, the conformational differences have been documented by changes in the apparent size of HSF, its sensitivity to denaturation in urea, and the facility with which a cysteine residue in the C-terminal domain can form crosslinks between monomers in the HSF trimer. it is poorly understood how the conformational state of HSF is regulated. Current evidence favors a feedback-regulation model, in which the equilibrium among HSF conformations is dependent on the chaperone, hsp70. Hsp70 is thought to facilitate the transition from the high-activity state to the low-activity state. This model, however, is unproven. It is furthermore unknown how HSF is activated, either during stress, or (at a lower level) during normal growth. The first main goal of this proposal is to obtain more precise information on the conformational changes that HSF undergoes, through a combination of functional assays and structural analyses. Transcriptional activator masking will be assessed through the affinity of HSF for general transcription factors. The general topology of hSF in its different conformations will be assessed through measurement of the accessibility of single cysteine residues introduced by site-directed mutagenesis. Interactions between specific domains of HSF will be assessed by induced crosslinking between cysteine residues. Physical constraints on critical residues in evolutionarily-conserved regions will be assessed by cassette mutagenesis. These approaches will help us describe the structure and function of HSF more completely, and should lead to an understanding of how the conformaitonal changes lead to activity changes. The second main goal of the proposal is to develop in vitro conditions for the activation and deactivation of HSF. This will enable athe biochemical analysis of these reactions, and allow the direct test of the hypothesis and hsp70 may be required to re-fold HSF to its low-activity state. The third main goal is to examine this system genetically, by (1) genetic screens for mutations suppressible by constitutive allels of hSF1, (2) genetic screens for mutations that display synthetic lethality with weak hsf1 alleles, and (3) screens for mutations or multicopy suppressors of genotypes that display a constitutive-heat-shock phenotype.
| 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 |
| Bonner, J J; Carlson, T; Fackenthal, D L et al. (2000) Complex regulation of the yeast heat shock transcription factor. Mol Biol Cell 11:1739-51 |
| 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 |
