Mecsas J, Cowing D W, Gross C A
Department of Bacteriology, University of Wisconsin-Madison 53706.
J Mol Biol. 1991 Aug 5;220(3):585-97. doi: 10.1016/0022-2836(91)90102-c.
We have charted the movements of E sigma 32 RNA polymerase at the heat-shock promoter PgroE throughout open complex formation, using hydroxyl radical footprinting. In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation. E sigma 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RPC1; in this complex the enzyme makes backbone contacts in the -35 region of the promoter that are maintained throughout open complex formation. An isomerization follows resulting in a second closed complex, RPC2; in this complex the enzyme makes base-specific and backbone contacts in the -10 region that are almost identical to those found in the open complex. Thus, at the groE promoter, upstream contacts are established in RPC1 and downstream contacts in RPC2. A similar pattern of backbone contacts was obtained for E sigma 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.
我们利用羟自由基足迹法，绘制了热休克启动子PgroE处E σ32 RNA聚合酶在整个开放复合物形成过程中的运动情况。结合甲基化保护和DNase I实验，这些数据为开放复合物的形成提出了以下模型。E σ32最初锚定在启动子的上游区域，形成第一个封闭复合物RPC1；在这个复合物中，酶在启动子的-35区域与主链接触，这种接触在整个开放复合物形成过程中一直保持。随后发生异构化，形成第二个封闭复合物RPC2；在这个复合物中，酶在-10区域与碱基特异性和主链接触，这些接触与开放复合物中的接触几乎相同。因此，在groE启动子处，上游接触在RPC1中建立，下游接触在RPC2中建立。在另外两个热休克启动子的开放复合物中结合的E σ32也获得了类似的主链接触模式，这表明无论启动子中的序列变化如何，开放复合物中全酶的整体拓扑结构是相似的。
