Roy Siddhartha, Semsey Szabolcs, Liu Mofang, Gussin Gary N, Adhya Sankar
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 2004 Nov 26;344(3):609-18. doi: 10.1016/j.jmb.2004.09.070.
GalR represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of GalR and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating GalR, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, GalR inhibited the slower of the two-step base-pair distortion process. We isolated a negative control GalR mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that GalR establishes contact with alpha-CTD at the last resolved isomerization intermediate, forming a kinetic trap.
GalR通过一种不依赖DNA环化的机制抑制galP1启动子。GalR和RNA聚合酶与DNA的平衡结合以及碱基对扭曲（异构化）的实时动力学表明，在饱和GalR存在的情况下，RNA聚合酶 - P1封闭复合物的平衡解离常数基本不受影响，这表明在该启动子处调节因子和RNA聚合酶之间不会发生相互拮抗（空间位阻）。在使用2 - AP标记的P1 DNA的荧光动力学实验中，GalR抑制了两步碱基对扭曲过程中较慢的那一步。我们分离出了一个阴性对照GalR突变体S29R，它虽然与操纵子DNA结合，但无法抑制P1。基于这些结果以及之前关于抑制需要RNA聚合酶α亚基的C末端结构域（α - CTD）的证明，我们提出GalR在最后解析的异构化中间体处与α - CTD建立接触，形成一个动力学陷阱。
