COOPERATIVE INTERACTION OF HUMAN HSF1 HEAT-SHOCK TRANSCRIPTION FACTORWITH PROMOTER DNA

We investigated the thermodynamics and kinetics of binding of human HS F1 heat shock transcription factor to different configurations of heat shock element (HSE) sequences on DNA fragments, in order to analyze b inding cooperativity under various conditions and to evaluate the sign ificance of interactions between multiple HSE sites. Constructs with d ifferent arrangements of one or more copies of a 15 base pair idealize d HSE sequence (AGAACGTTCTAGAAC) were used for in vitro binding experi ments performed by multiple probe band shift assays and titrations. Di ssociation kinetics under various conditions were also measured by ban d shift assays. These experiments indicated significant differences in behavior between constructs with a pair of tandem sites in correct or ientation (forming a continuous array of alternating GAA and TTC block s), and those with only a single site, or a pair of sites in reversed orientation. These differences in behavior indicated significant effec ts of cooperative binding to tandem sites in vitro, and showed in part icular a strong temperature dependence of binding to different constru cts. Thermodynamic parameters for binding affinity and cooperativity w ere also evaluated from direct titrations.