Hwang Candy S, Xu Liang, Wang Wei, Ulrich Sébastien, Zhang Lu, Chong Jenny, Shin Ji Hyun, Huang Xuhui, Kool Eric T, McKenna Charles E, Wang Dong
Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA.
Department of Cellular and Molecular Medicine, School of Medicine; Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
Nucleic Acids Res. 2016 May 5;44(8):3820-8. doi: 10.1093/nar/gkw220. Epub 2016 Apr 7.
RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous 'synthetic nucleic acid substitution' strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-'substituted' NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the β,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.
RNA聚合酶II（pol II）利用一个复杂的相互作用网络来高效且准确地选择并掺入正确的核苷三磷酸（NTP）底物。我们之前的“合成核酸替代”策略已成功应用于剖析pol II转录中核酸部分的功能。然而，底物的三磷酸部分如何影响核苷酸掺入过程中P-O键的断裂和形成速率仍不清楚。在此，通过使用β,γ-桥连原子“取代”的NTP，我们阐明了焦磷酸离去基团中的亚甲基取代如何影响同源和非同源核苷酸的掺入。有趣的是，β,γ-亚甲基取代对非同源UTP/dT支架的影响（kpol降低约3倍）与同源ATP/dT支架的影响（kpol降低约130倍）显著不同。去除U:dT中的摆动氢键可恢复对UTP亚甲基取代的强烈响应。我们的动力学和建模研究与UTP/dT掺入相比ATP/dT掺入时独特的键形成和断裂过渡态变化一致。总体而言，我们的数据揭示了核苷酸掺入过程中NTP三磷酸部分与碱基对氢键识别之间的功能相互作用。
