Wang Y, Morgan W D
Department of Biology, McGill University, Montreal, Quebec, Canada.
Nucleic Acids Res. 1994 Aug 11;22(15):3113-8. doi: 10.1093/nar/22.15.3113.
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.
我们研究了人类HSF1热休克转录因子与DNA片段上热休克元件（HSE）序列的不同构型结合的热力学和动力学，以便分析各种条件下的结合协同性，并评估多个HSE位点之间相互作用的重要性。通过多个探针带迁移分析和滴定法进行体外结合实验，使用了具有一个或多个15个碱基对理想化HSE序列（AGAACGTTCTAGAAC）拷贝的不同排列的构建体。还通过带迁移分析测量了各种条件下的解离动力学。这些实验表明，具有一对正确取向的串联位点（形成交替的GAA和TTC块的连续阵列）的构建体与仅具有单个位点或一对反向位点的构建体之间在行为上存在显著差异。这些行为差异表明体外与串联位点的协同结合具有显著影响，尤其显示出与不同构建体结合的强烈温度依赖性。还通过直接滴定评估了结合亲和力和协同性的热力学参数。
