The DNA-binding domain of yeast heat shock transcription factor independently regulates both the N- and C-terminal activation domains.
The expression of heat shock proteins in response to cellular stresses is dependent on the activity of the heat shock transcription factor (HSF). In yeast, HSF is constitutively bound to DNA; however, the mitigation of negative regulation in response to stress dramatically increases transcriptional activity. Through alanine-scanning mutagenesis of the surface residues of the DNA-binding domain, we have identified a large number of mutants with increased transcriptional activity. Six of the strongest mutations were selected for detailed study. Our studies suggest that the DNA-binding domain is involved in the negative regulation of both the N-terminal and C-terminal activation domains of HSF. These mutations do not significantly affect DNA binding. Circular dichroism analysis suggests that a subset of the mutants may have altered secondary structure, whereas a different subset has decreased thermal stability. Our findings suggest that the regulation of HSF transcriptional activity (under both constitutive and stressed conditions) may be partially dependent on the local topology of the DNA-binding domain. In addition, the DNA-binding domain may mediate key interactions with ancillary factors and/or other intramolecular regulatory regions in order to modulate the complex regulation of HSF's transcriptional activity.
Bulman
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Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 422 Curie Blvd., Philadelphia, PA 19104, USA.
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Nelson
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007716:Kluyveromyces / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020218:Response Elements; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C067168:HSF1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors