Departments of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15121-6. doi: 10.1073/pnas.1208508109. Epub 2012 Sep 4.
Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu(+)-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.
金属调控因子通过响应金属离子来调节转录。许多研究已经深入了解了 MerR 家族金属调控因子在金属结合后如何激活转录。相比之下，激活后转录如何关闭尚不清楚。然而，迅速关闭转录非常重要，因为细胞不希望在解除金属压力后继续表达金属抗性基因，从而浪费能量。我们使用单分子 FRET 测量研究了铜外排调节剂（CueR）与 DNA 的动态相互作用，CueR 是一种对 Cu(+）有反应的 MerR 家族金属调控因子。除了定量其 DNA 结合和解离动力学外，我们还发现 CueR 在识别位点处自发翻转其结合取向。CueR 还有两种不同的结合模式，分别对应于与特定和非特异性 DNA 序列的相互作用，这将有助于识别定位。最引人注目的是，来自溶液中的 CueR 分子可以直接替代 DNA 结合的 CueR 或协助在位 CueR 的解离，这两种情况在任何 DNA 结合蛋白中都是独特的例子。直接蛋白质替代和辅助解离反应的动力学表明，这两个独特的过程可以提供有效的途径，用 apo-CueR 替代 DNA 结合的 holo-CueR，从而迅速而轻松地关闭转录。
