College of Chemistry and Materials Science, Sichuan Normal University, Chengdu, China.
College of Resources and Environment, Chengdu University of Information Technology, Chengdu, China.
Biophys J. 2018 Apr 24;114(8):1755-1761. doi: 10.1016/j.bpj.2018.02.033.
RNA polymerase (RNAP) is the primary machine responsible for transcription. Its ability to distinguish between correct (cognate) and incorrect (noncognate) nucleoside triphosphates (NTPs) is important for fidelity control in transcription. In this work, we investigated the substrate selection mechanism of T7 RNAP from the perspective of energetics. The dissociation free energies were determined for matched and unmatched base pairs in the preinsertion complex using the umbrella sampling method. A clear hydrogen-bond-rupture peak is observed in the potential of mean force curve for a matched base pair, whereas no such peaks are present in the position of mean force profiles for unmatched ones. The free-energy barrier could prevent correct substrates from being separated from the active site. Therefore, when NTPs diffuse into the active site, correct ones will stay for chemistry once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution. This result provides an important energy evidence for the substrate selection mechanism of RNAP. Then we elucidated energetics and molecular details for correct NTP binding to the active site of the insertion complex. Our observations reveal that strong interactions act on the triphosphate of NTP to constrain its movement, whereas relatively weak interactions serve to position the base in the correct conformation. Triple interactions, hydrophobic contacts from residues M635 and Y639, base stacking from the 3' RNA terminal nucleotide, and base pairing from the template nucleotide act together to position the NTP base in a catalytically competent conformation. At last, we observed that incorrect NTPs cannot be as well-stabilized as the correct one in the active site when they are misincorporated in the insertion site. It is expected that our work can be helpful for comprehensively understanding details of this basic step in genetic transcription.
RNA 聚合酶 (RNAP) 是负责转录的主要机器。它区分正确（同源）和不正确（非同源）核苷三磷酸 (NTP) 的能力对于转录保真度控制很重要。在这项工作中，我们从能量学的角度研究了 T7 RNAP 的底物选择机制。使用伞状采样法确定了预插入复合物中匹配和不匹配碱基对的离解自由能。在匹配碱基对的平均力曲线的势能中观察到明显的氢键断裂峰，而在不匹配碱基对的平均力曲线中没有这样的峰。自由能势垒可以防止正确的底物与活性位点分离。因此，当 NTP 扩散到活性位点时，如果与模板核苷酸建立有效的碱基配对接触，正确的 NTP 将在化学上停留，而不正确的 NTP 将从活性位点撤出并被拒绝回到溶液中。这一结果为 RNAP 的底物选择机制提供了重要的能量证据。然后，我们阐明了正确的 NTP 与插入复合物的活性位点结合的能量学和分子细节。我们的观察结果表明，强相互作用作用于 NTP 的三磷酸基团，以限制其运动，而相对较弱的相互作用则将碱基定位在正确的构象中。三键相互作用、来自残基 M635 和 Y639 的疏水性接触、3'RNA 末端核苷酸的碱基堆积和模板核苷酸的碱基配对共同将 NTP 碱基定位在催化活性构象中。最后，我们观察到，当插入位点错误掺入时，不正确的 NTP 不能像正确的 NTP 那样在活性位点中得到很好的稳定。预计我们的工作可以帮助全面理解遗传转录这一基本步骤的细节。
