Da Lin-Tai, E Chao, Duan Baogen, Zhang Chuanbiao, Zhou Xin, Yu Jin
Department of Physics and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
Beijing Computational Science Research Center, Beijing, China.
PLoS Comput Biol. 2015 Nov 24;11(11):e1004624. doi: 10.1371/journal.pcbi.1004624. eCollection 2015 Nov.
Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase.
转录延伸过程中焦磷酸离子（PPi）的释放是每个核苷酸添加循环中的标志性步骤。该过程的动力学和能量学，以及它如何随着聚合酶复合物的大量构象变化而进行，决定了转录延伸的机械化学偶联机制。在这里，我们通过实施全原子分子动力学（MD）模拟，研究了噬菌体T7单亚基RNA聚合酶（RNAP）中PPi释放过程的详细动力学。我们利用广泛的纳秒MD模拟获得了PPi释放的跳出腔动力学模型。我们发现T7 RNAP中的PPi释放是由PPi从两个催化天冬氨酸解离开始的，随后是一个相对缓慢的跳出腔激活过程。结合多个微秒级的长MD模拟，我们还发现激活过程受到带电残基缔合以及局部空间位阻和氢键相互作用的阻碍。另一方面，一个高度灵活的赖氨酸残基Lys472通过摆动其侧链将PPi拉出，极大地促进了激活。该机制通常适用于具有相似分子结构和保守关键残基的单亚基RNA和DNA聚合酶。值得注意的是，即使在具有电荷促进跳跃机制的多亚基RNAP中，灵活的赖氨酸或精氨酸残基似乎也是辅助PPi释放的通用模块。我们还通过微秒MD模拟注意到，PPi释放与T7 RNAP手指结构域上O螺旋的打开运动没有紧密耦合。因此，我们的研究支持了单亚基聚合酶转录延伸中机械化学偶联的布朗棘轮模型。
