GENETIC IDENTIFICATION OF THE SITE OF DNA CONTACT IN THE YEAST HEAT-SHOCK TRANSCRIPTION FACTOR

The heat shock transcription factor (HSF), a trimeric transcription fa ctor, activates the expression of heat shock genes in eukaryotes, We h ave isolated mutations in the HSF1 gene from Saccharomyces cerevisiae that severely compromise the ability of HSF to bind to its normal bind ing site, repeats of the module nGAAn. One of these mutations, Q229R, shows a ''new specificity'' phenotype, in which the protein prefers th e mutant sequence nGACn. These results identify the region of HSF that contacts DNA, in complete agreement with the crystal structure of HSF of Kluyveromyces lactis and the nuclear magnetic resonance data from HSF of Drosophila melanogaster. To determine the orientation of the DN A-binding domain on the nGAAn motif, we performed site-specific cross- linking between cysteine residues of single cysteine substitutions. Cy steines placed at the N terminus of the DNA contact helix formed cross -links readily, while cysteines placed at the C terminus of the helix did not.