Zhang X, Reeder T, Schleif R
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
J Mol Biol. 1996 Apr 26;258(1):14-24. doi: 10.1006/jmbi.1996.0230.
We studied the formation of open complexes of RNA polymerase and promoter DNA as activated by the AraC protein at the Escherichia coli araBAD promoter pBAD and by the cyclic AMP receptor protein at the galKTE promoter P1. The DNA migration retardation assay was demonstrated to be suitable for the detection and quantitation of open complexes by the correspondence in the properties of open complexes in solution and retarded complexes observed in gels. These included, on the ara promoter, heparin resistance, lifetime, DNAseI footprinting, exonuclease III footprinting, permanganate footprinting and disappearance upon transcription, and on the gal promoter, the correspondence between the kinetic parameters Kd and k2 obtained with established techniques and those obtained with the migration retardation assay. On the pBAD promoter we obtained kinetic parameters of Kd = 0.3 nM and K2 = 1 minute(-1). The unusually tight binding of polymerase in the presence of AraC suggests that AraC binds polymerase tightly.
我们研究了在大肠杆菌araBAD启动子pBAD处由AraC蛋白激活以及在galKTE启动子P1处由环磷酸腺苷受体蛋白激活时，RNA聚合酶与启动子DNA开放复合物的形成。通过溶液中开放复合物的性质与凝胶中观察到的滞后复合物之间的对应关系，证明DNA迁移阻滞试验适用于开放复合物的检测和定量。这些性质包括在ara启动子上的肝素抗性、寿命、DNA酶I足迹、核酸外切酶III足迹、高锰酸盐足迹以及转录时的消失，以及在gal启动子上，用既定技术获得的动力学参数Kd和k2与用迁移阻滞试验获得的参数之间的对应关系。在pBAD启动子上，我们获得的动力学参数为Kd = 0.3 nM和K2 = 1分钟-1。在AraC存在的情况下，聚合酶异常紧密的结合表明AraC与聚合酶紧密结合。
