Ceglarek J A, Revzin A
Department of Biochemistry, Michigan State University, East Lansing 48824-1319.
Electrophoresis. 1989 May-Jun;10(5-6):360-5. doi: 10.1002/elps.1150100514.
The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.
在核酸 - 蛋白质（尤其是DNA - 蛋白质）相互作用的研究中，凝胶电泳的应用已经产生了关于多种此类系统的大量定性和定量信息。与游离DNA相比，复合物迁移率的降低使得复合物能够被分离和表征，同时也能确定相互作用的热力学和动力学性质。本文首先回顾了“凝胶阻滞”分析的近期应用，以此作为对两个领域实验的介绍。在第一个领域中，测试了一个假设：两端附近结合了大量蛋白质的DNA分子在聚丙烯酰胺凝胶中的迁移方式是否与蛋白质位于DNA片段中间的相应复合物不同。数据显示，这些类型的复合物迁移率差异很小，这意味着两者可能以线性的“蛇形”方式在凝胶中移动。这些实验还提供了与大肠杆菌分解代谢物激活蛋白（CAP）和RNA聚合酶结合到乳糖启动子区域所导致的DNA弯曲问题相关的结果。似乎在野生型lac结合位点处由CAP引起的DNA弯曲在RNA聚合酶同时结合在lac UV5启动子的复合物中得以保留。
