Brenowitz M, Senear D F, Shea M A, Ackers G K
Proc Natl Acad Sci U S A. 1986 Nov;83(22):8462-6. doi: 10.1073/pnas.83.22.8462.
A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.
基因调控中的一个核心问题是调节蛋白配体与DNA上特定位点协同结合的机制及生物学功能。为阐明这些相互作用的物理化学基础，我们开发了一种用于进行DNase I“足迹”（保护）滴定实验的热力学严谨方法。通过该技术测定的单个位点结合曲线，可以解析出内在结合常数以及不同位点之间协同相互作用的常数。对cI阻遏物 - 操纵基因结合的实验研究表明，该方法能够准确反映结合位点的分数饱和度。我们给出了具有两个活性位点的λ操纵基因的单个位点结合曲线，证明了位点之间存在协同相互作用。这些曲线设定了协同自由能大小的下限，而无需与单一位点突变操纵基因进行比较。
