The major goal of the proposed research is to understand how proteins specifically recognize DNA and how this interaction modulates transcriptional regulation. Heat shock transcription factor (HSF) has been chosen for study because of its important biological role and unique structura features. HSF is the eukaryotic transcriptional activator for the heat shock response, an evolutionarily conserved mechanism which protects cells from heat and other environmental perturbations. This response is also invoked by developmental and physiological stimuli. HSF is found in all eukaryotes, from yeast to humans. Yeast and fruitflies have a single HSF, while more complex eukaryotes have multiple HSFs, which respond to different stresses and exhibit differential gene expression. HSF is one of only a few examples of a sequence-specific, homotrimeric DNA binding protein, and the timeric nature of HSF poses interesting questions about how a three-fold symmetric protein can bind to DNA. In addition to binding as a trimer, HSF has multiple layers of cooperativity involved with it DNA binding, and this cooperativity has functional implications for promoter selectivity in vivo. All HSFs recognize the same DNA sequence, called a heat shock element (HSE), and have a common, conserved core that includes the DNA binding and trimerization domains. Fragments of HSF containing just this core have the same DNA Binding and oligomerization properties as full length HSF. These fragments are highly amenable to physical studies, and yeast HSF is amenable to genetic approaches. Because of the conservation of HSFs, studies o yeast HSF are directly applicable to human HSFs. The key to the proposed approach is the interplay between biochemical, crystallographic, and genetic studies.
The specific aims of this proposal are to: understand the basis of HSF's specificity to its DNA binding site by focusing on the DNA binding domain-HSE interaction; characterize the functiona role of two unusual structural features that are located within the DNA bindin domain and are postulated to be involved in cooperativity; and determine the structural accommodations that a three-fold symmetric, trimeric protein must make to bind DNA. These experiments will deepen our understanding of how HSF binds to HSEe and will eventually lead to the long term goal of being able to manipulate HSF's function in order to take advantage of its protective role.
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