Wu Shaogui, Li Laicai, Li Quan
College of Chemistry and Materials Science, Sichuan Normal University, Chengdu, China; State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, Beijing, China.
College of Chemistry and Materials Science, Sichuan Normal University, Chengdu, China.
Biophys J. 2017 Jun 6;112(11):2253-2260. doi: 10.1016/j.bpj.2017.04.039.
In genetic transcription, molecular dynamic details and energetics of NTP binding to the active site of RNA polymerase (RNAP) are poorly understood. In this article, we investigated the NTP binding process in T7 RNAP using all-atom MD simulation combined with the umbrella sampling technique. Based on our simulations, a two-step mechanism was proposed to explain NTP binding: first, substrate NTP in aqueous solution, which carries a magnesium ion, diffuses through a secondary channel of RNAP to attain a pore region, where it undergoes conformational changes to give a correct orientation; next, the NTP establishes initial basepairing contacts with the template nucleoside (TN). Our free-energy calculations suggest that both steps are spontaneous. This mechanism can easily explain the problem of NTP binding with different orientations. Moreover, it is found that the nascent NTP:TN basepair is fragile and easily broken by thermal disturbance. Therefore, we speculate that the fingers domain will be triggered to close, so as to create a steady environment for the next chemical step. The observations from the work provide valuable information for comprehensively understanding the mechanism of the basic step in genetic transcription.
在基因转录过程中，人们对三磷酸核苷（NTP）与RNA聚合酶（RNAP）活性位点结合的分子动力学细节和能量学了解甚少。在本文中，我们结合伞形抽样技术，利用全原子分子动力学（MD）模拟研究了T7 RNA聚合酶中的NTP结合过程。基于我们的模拟，提出了一种两步机制来解释NTP结合：首先，携带镁离子的水溶液中的底物NTP通过RNAP的二级通道扩散到一个孔区域，在那里它发生构象变化以获得正确的方向；接下来，NTP与模板核苷（TN）建立初始碱基配对接触。我们的自由能计算表明这两个步骤都是自发的。该机制可以轻松解释NTP以不同方向结合的问题。此外，发现新生的NTP:TN碱基对很脆弱，容易受到热干扰而断裂。因此，我们推测手指结构域将被触发关闭，以便为下一个化学步骤创造一个稳定的环境。这项工作的观察结果为全面理解基因转录基本步骤的机制提供了有价值的信息。
