Wang Beibei, Opron Kristopher, Burton Zachary F, Cukier Robert I, Feig Michael
Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
Nucleic Acids Res. 2015 Jan;43(2):1133-46. doi: 10.1093/nar/gku1370. Epub 2014 Dec 30.
Transcriptional fidelity, which prevents the misincorporation of incorrect nucleoside monophosphates in RNA, is essential for life. Results from molecular dynamics (MD) simulations of eukaryotic RNA polymerase (RNAP) II and bacterial RNAP with experimental data suggest that fidelity may involve as many as five checkpoints. Using MD simulations, the effects of different active site NTPs in both open and closed trigger loop (TL) structures of RNAPs are compared. Unfavorable initial binding of mismatched substrates in the active site with an open TL is proposed to be the first fidelity checkpoint. The leaving of an incorrect substrate is much easier than a correct one energetically from the umbrella sampling simulations. Then, the closing motion of the TL, required for catalysis, is hindered by the presence of mismatched NTPs. Mismatched NTPs also lead to conformational changes in the active site, which perturb the coordination of magnesium ions and likely affect the ability to proceed with catalysis. This step appears to be the most important checkpoint for deoxy-NTP discrimination. Finally, structural perturbations in the template DNA and the nascent RNA in the presence of mismatches likely hinder nucleotide addition and provide the structural foundation for backtracking followed by removing erroneously incorporated nucleotides during proofreading.
转录保真度可防止RNA中错误的核苷单磷酸误掺入，对生命至关重要。真核生物RNA聚合酶（RNAP）II和细菌RNAP的分子动力学（MD）模拟结果与实验数据表明，保真度可能涉及多达五个检查点。利用MD模拟，比较了不同活性位点NTP在RNAP开放和闭合触发环（TL）结构中的作用。活性位点中错配底物与开放TL的不利初始结合被认为是第一个保真度检查点。从伞形抽样模拟来看，错误底物从能量角度比正确底物更容易离开。然后，催化所需的TL闭合运动受到错配NTP存在的阻碍。错配NTP还会导致活性位点的构象变化，从而扰乱镁离子的配位，并可能影响催化进行的能力。这一步似乎是脱氧NTP识别的最重要检查点。最后，错配情况下模板DNA和新生RNA中的结构扰动可能会阻碍核苷酸添加，并为校对过程中回溯并去除错误掺入的核苷酸提供结构基础。
