A prehydrolysis state of an AAA plus ATPase supports transcription activation of an enhancer-dependent RNA polymerase
A prehydrolysis state of an AAA plus ATPase supports transcription activation of an enhancer-dependent RNA polymerase
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DOI：
10.1073/pnas.1001188107
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发表时间：
2010-05-18
影响因子：
11.1
通讯作者：
Buck, Martin
中科院分区：
文献类型：
--
作者：
Burrows, Patricia C.;Joly, Nicolas;Buck, Martin
ATP水解依赖的分子机器和马达通常驱动细胞信号传导和基因调控复合物中的受调控构象转化。含有细菌RNA聚合酶（RNAP）主要变体形式E sigma（54）的基因调控复合物的构象重组需要与其同源ATP水解激活蛋白结合。重要的是，这种激活的RNAP对于许多适应性反应是必不可少的，包括细菌致病所需的那些。在这里，我们使用小引物RNA（spRNA）合成测定来表征增强子依赖性E sigma（54）和其同源激活剂AAA+ ATP酶蛋白在ADP+P（i）形成之前的初始相遇。结果表明，在预水解状态下，E sigma（54）中发生了足够的激活剂依赖性重排，使RNAP活性位点与单链启动子DNA接合，以支持spRNA合成，但不使启动子DNA熔化。这种具有催化活性的转录中间体与开放启动子复合物具有相似性，因为DNA卷曲所需的RNAP动力学应该发生。值得注意的是，这项工作强调了ATP酶的预水解状态在它们驱动的分子转化中具有重要的功能。
ATP hydrolysis-dependent molecular machines and motors often drive regulated conformational transformations in cell signaling and gene regulation complexes. Conformational reorganization of a gene regulation complex containing the major variant form of bacterial RNA polymerase (RNAP), E sigma(54), requires engagement with its cognate ATP-hydrolyzing activator protein. Importantly, this activated RNAP is essential for a number of adaptive responses, including those required for bacterial pathogenesis. Here we characterize the initial encounter between the enhancer-dependent E sigma(54) and its cognate activator AAA+ ATPase protein, before ADP+P(i) formation, using a small primed RNA (spRNA) synthesis assay. The results show that in a prehydrolysis state, sufficient activator-dependent rearrangements in E sigma(54) have occurred to allow engagement of the RNAP active site with single-stranded promoter DNA to support spRNA synthesis, but not to melt the promoter DNA. This catalytically competent transcription intermediate has similarity with the open promoter complex, in that the RNAP dynamics required for DNA scrunching should be occurring. Significantly, this work highlights that prehydrolysis states of ATPases are functionally important in the molecular transformations they drive.