Das Ranabir, Loss Sandra, Li Jess, Waugh David S, Tarasov Sergey, Wingfield Paul T, Byrd R Andrew, Altieri Amanda S
Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
J Mol Biol. 2008 Feb 22;376(3):705-20. doi: 10.1016/j.jmb.2007.11.022. Epub 2007 Nov 17.
In prokaryotic transcription regulation, several host factors form a complex with RNA polymerase and the nascent mRNA. As part of a process known as antitermination, two of these host factors, NusB and NusE, bind to form a heterodimer, which interacts with a specific boxA site on the RNA. The NusB/NusE/boxA RNA ternary complex interacts with the RNA polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. The NusB protein also binds boxA RNA individually and retains all specificity for boxA. However, NusE increases the affinity of RNA to NusB in the ternary complex, which contributes to efficient antitermination. To understand the molecular mechanism of the process, we have determined the structure of NusB from the thermophilic bacterium Aquifex aeolicus and studied the interaction of NusB and NusE. We characterize this binding interaction using NMR, isothermal titration calorimetry, gel filtration, and analytical ultracentrifugation. The binding site of NusE on NusB was determined using NMR chemical shift perturbation studies. We have also determined the NusE binding site in the ternary Escherichia coli NusB/NusE/boxA RNA complex and show that it is very similar to that in the NusB/NusE complex. There is one loop of residues (from 113 to 118 in NusB) affected by NusE binding in the ternary complex but not in the binary complex. This difference may be correlated to an increase in binding affinity of RNA for the NusB/NusE complex.
在原核生物转录调控中，几种宿主因子与RNA聚合酶和新生mRNA形成复合物。作为抗终止过程的一部分，其中两种宿主因子NusB和NusE结合形成异源二聚体，该异源二聚体与RNA上的特定boxA位点相互作用。NusB/NusE/boxA RNA三元复合物与RNA聚合酶转录复合物相互作用，使其稳定并允许转录越过过早终止点。NusB蛋白也可单独结合boxA RNA，并保留对boxA的所有特异性。然而，NusE增加了三元复合物中RNA与NusB的亲和力，这有助于高效抗终止。为了理解该过程的分子机制，我们测定了嗜热菌嗜热栖热菌的NusB结构，并研究了NusB和NusE的相互作用。我们使用核磁共振、等温滴定量热法、凝胶过滤和分析超速离心来表征这种结合相互作用。利用核磁共振化学位移扰动研究确定了NusE在NusB上的结合位点。我们还确定了大肠杆菌NusB/NusE/boxA RNA三元复合物中NusE的结合位点，并表明它与NusB/NusE复合物中的结合位点非常相似。在三元复合物中有一个残基环（NusB中的113至118位）受NusE结合的影响，但在二元复合物中不受影响。这种差异可能与RNA对NusB/NusE复合物结合亲和力的增加有关。
