A GEL-ELECTROPHORESIS METHOD FOR QUANTIFYING THE BINDING OF PROTEINS TO SPECIFIC DNA REGIONS - APPLICATION TO COMPONENTS OF THE ESCHERICHIA-COLI LACTOSE OPERON REGULATORY SYSTEM
A GEL-ELECTROPHORESIS METHOD FOR QUANTIFYING THE BINDING OF PROTEINS TO SPECIFIC DNA REGIONS - APPLICATION TO COMPONENTS OF THE ESCHERICHIA-COLI LACTOSE OPERON REGULATORY SYSTEM
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DOI：
10.1093/nar/9.13.3047
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发表时间：
1981-01-01
影响因子：
14.9
通讯作者：
REVZIN, A
中科院分区：
文献类型：
--
作者：
GARNER, MM;REVZIN, A
描述了使用凝胶电泳定量研究 DNA-蛋白质相互作用。这种快速而简单的技术涉及根据 DNA-蛋白质复合物在聚丙烯酰胺凝胶中电泳迁移率的差异将游离 DNA 从它们中分离出来。在有利的条件下，可以对未结合的DNA和与蛋白质相关的DNA进行定量。该凝胶方法应用于大肠杆菌的研究。乳糖操纵子调节系统。在生理范围内的离子强度下，分解代谢物激活蛋白 (CAP) 与野生型 lac 启动子形成长寿命复合物，但不与 CAP 不敏感突变体形成。 RNA聚合酶与野生型启动子形成稳定的“开放”或“熔入”复合物需要CAP和环AMP的参与。此外，还证明，即使在 CAP-cAMP 存在下预形成，如果随后选择性去除 CAP，聚合酶-启动子开放复合物也会变得不稳定。
The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified.This gel method is applied to the study of theE. colilactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild typelacpromoter, but not with a CAP-insensitive mutant. Formation of a stable “open” or “melted-in” complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.