Cowing D W, Mecsas J, Record M T, Gross C A
Department of Bacteriology, University of Wisconsin-Madison 53706.
J Mol Biol. 1989 Dec 5;210(3):521-30. doi: 10.1016/0022-2836(89)90128-9.
The interaction of E sigma 32 with the groE promoter at temperatures between 0 degrees C and 37 degrees C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 degrees C E sigma 32 fully protected sequences between -60 and -5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 degrees C the majority of the E sigma 32 promoter complexes had a DNase I footprint almost identical with that seen at 37 degrees C, protecting the DNA from about -60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by E sigma 32 differed from those seen at 37 degrees C. DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 degrees C and 27 degrees C. It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 degrees C. Therefore we propose that the formation of an open complex by E sigma 32 at the groE promoter involves three classes of steps: E sigma 32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex. E sigma 32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex. Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO).
利用DNA酶I足迹法和硫酸二甲酯甲基化法研究了在0℃至37℃之间的温度下E σ32与groE启动子的相互作用。观察到三种不同的复合物。在0℃时，E σ32完全保护启动子上链（非模板链）上-60至-5之间的序列不被DNA酶I消化。在16℃时，大多数E σ32启动子复合物的DNA酶I足迹与在37℃时观察到的几乎相同，保护DNA从约-60至+20；然而，几乎没有发生DNA链分离，并且E σ32引起的鸟嘌呤残基对硫酸二甲酯甲基化敏感性的变化与在37℃时观察到的不同。在16℃至27℃之间的温度下发生了DNA链分离，以及硫酸二甲酯对甲基化的保护和增强模式向开放复合物特征模式的变化。可以合理地假设这些温度依赖性异构化类似于在37℃时开放复合物形成途径上中间体的时间依赖性序列。因此，我们提出E σ32在groE启动子上形成开放复合物涉及三类步骤：E σ32最初以封闭复合物（RPC1）结合到启动子上，其中酶与DNA的较小区域相互作用，比在开放复合物中相互作用的区域小。然后E σ32异构化形成第二个封闭复合物（RPC2），其中酶与开放复合物中相同的DNA区域相互作用。最后，局部DNA变性（链开放）过程导致开放复合物（RPO）的形成。
