Open AccessGenetic Identification of the Site of DNA Contact in the Yeast Heat Shock Transcription Factor
Author(s) -
Fernando Araripe Gonçalves Torres,
J. José Bonner
Publication year - 1995
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.15.9.5063
Subject(s) - biology , heat shock factor , yeast , transcription factor , genetics , dna binding site , dna , identification (biology) , promoter , heat shock protein , gene , hsp70 , gene expression , botany
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.