Cooperative interaction of human HSF1 heat shock transcription factor with promoter DNA.
We investigated the thermodynamics and kinetics of binding of human HSF1 heat shock transcription factor to different configurations of heat shock element (HSE) sequences on DNA fragments, in order to analyze binding cooperativity under various conditions and to evaluate the significance of interactions between multiple HSE sites. Constructs with different arrangements of one or more copies of a 15 base pair idealized HSE sequence (AGAACGTTCTAGAAC) were used for in vitro binding experiments performed by multiple probe band shift assays and titrations. Dissociation kinetics under various conditions were also measured by band shift assays. These experiments indicated significant differences in behavior between constructs with a pair of tandem sites in correct orientation (forming a continuous array of alternating GAA and TTC blocks), and those with only a single site, or a pair of sites in reversed orientation. These differences in behavior indicated significant effects of cooperative binding to tandem sites in vitro, and showed in particular a strong temperature dependence of binding to different constructs. Thermodynamic parameters for binding affinity and cooperativity were also evaluated from direct titrations.
Wang
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Department of Biology, McGill University, Montreal, Quebec, Canada.
Morgan
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D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015636:Magnesium Chloride; D004247:DNA; D005982:Glutathione Transferase