Genetic identification of the site of DNA contact in the yeast heat shock transcription factor.
The heat shock transcription factor (HSF), a trimeric transcription factor, activates the expression of heat shock genes in eukaryotes. We have isolated mutations in the HSF1 gene from Saccharomyces cerevisiae that severely compromise the ability of HSF to bind to its normal binding site, repeats of the module nGAAn. One of these mutations, Q229R, shows a "new specificity" phenotype, in which the protein prefers the 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 DNA-binding domain on the nGAAn motif, we performed site-specific cross-linking between cysteine residues of single-cysteine substitutions. Cysteines 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.
Torres
F A
FA
Department of Biology, Indiana University, Bloomington 47405, USA.
Bonner
J J
JJ
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D006360:Heat-Shock Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C067168:HSF1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors