Carey J
Department of Biochemistry, Stanford University Medical Center, CA 94305-5307.
Proc Natl Acad Sci U S A. 1988 Feb;85(4):975-9. doi: 10.1073/pnas.85.4.975.
The affinity and stoichiometry of DNA binding by Escherichia coli trp repressor were studied by electrophoresis in nondenaturing gels. The ability of trp repressor to retard the electrophoretic mobility of an operator DNA fragment depends on the pH of the gel system. Above the pI of the protein, little retardation of DNA is observed, although complex formation can be detected by other assays. As the pH of the gel is lowered, retardation is enhanced. The apparent dissociation constant for the interaction between trp repressor and trpEDCBA operator fragments is 0.5 nM under the conditions used here. Nonspecific binding occurs with only about 200-fold weaker affinity. The stoichiometries of specific and nonspecific complexes were determined directly by using trp repressor labeled in vivo. High-affinity operator binding requires a single dimer of trp repressor. DNase I-protection analysis ("footprinting") was used to confirm the dissociation constants and to locate the binding site.
通过非变性凝胶电泳研究了大肠杆菌色氨酸阻遏物与DNA结合的亲和力和化学计量关系。色氨酸阻遏物使操纵基因DNA片段电泳迁移率减慢的能力取决于凝胶系统的pH值。在蛋白质的等电点以上，尽管可以通过其他检测方法检测到复合物的形成，但观察到DNA的迁移率几乎没有减慢。随着凝胶pH值的降低，迁移率减慢增强。在此处使用的条件下，色氨酸阻遏物与trpEDCBA操纵基因片段之间相互作用的表观解离常数为0.5 nM。非特异性结合的亲和力仅约弱200倍。通过使用体内标记的色氨酸阻遏物直接测定了特异性和非特异性复合物的化学计量关系。高亲和力的操纵基因结合需要一个色氨酸阻遏物二聚体。使用DNase I保护分析（“足迹法”）来确认解离常数并定位结合位点。
