A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation.
A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation.
复制标题
T7 RNA 聚合酶转录延伸中关键赖氨酸残基协助从腔跳跃释放焦磷酸离子
DOI：
10.1371/journal.pcbi.1004624
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发表时间：
2015-11
影响因子：
4.3
通讯作者：
Yu J
中科院分区：
文献类型：
--
作者：
Da LT;E C;Duan B;Zhang C;Zhou X;Yu J
焦磷酸离子(PPI)在转录延伸过程中的释放是每个核苷酸加成循环中的一个标志性步骤。这个过程的动力学和能量学以及它是如何随着聚合酶复合体的实质性构象变化而进行的，决定了转录延伸的机械力-化学耦合机制。在这里，我们研究了来自噬菌体T7的单亚单位RNA聚合酶(RNAP)中PPI释放过程的详细动力学，实现了全原子分子动力学(MD)模拟。我们利用广泛的纳秒分子动力学模拟得到了PPI释放的空腔跳跃动力学模型。我们发现，T7RNAP中PPI的释放是由两个催化天冬氨酸的PPI解离引发的，随后是一个相对缓慢的从空腔跳跃的激活过程。结合一些微秒长的分子动力学模拟，我们还发现，活化过程受到带电残基缔合以及局部空间和氢键相互作用的阻碍。另一方面，高度灵活的赖氨酸残基Lys472可以摆动侧链将PPI拉出，从而极大地帮助激活。该机制一般适用于分子结构相似、关键残基保守的单亚基RNA和DNA聚合酶。值得注意的是，灵活的赖氨酸或精氨酸残基似乎是一个通用的模块，即使在具有电荷促进跳跃机制的多亚基RNAP中也能帮助PPI的释放。我们还注意到，根据微秒MD模拟，PPI的释放与T7 RNAP指状域上O-螺旋的打开运动没有紧密耦合。因此，我们的研究支持单亚基聚合酶转录延伸中机械力-化学耦合的布朗棘轮假说。RNA聚合酶(RNAP)通常被认为是布朗棘轮机器，在转录延伸过程中机械运动和化学反应松散耦合。然而，对单亚单位T7 RNAP的结构研究提出了一种替代的力量卒中情景，这需要PPI产物释放反应与聚合酶易位紧密结合或驱动。为了解决机械力-化学耦合情景中相互矛盾的观点，我们进行了原子分子动力学(MD)模拟，研究了T7 RNAP的PPI释放机制。利用大量的纳秒短模拟，我们构建了马尔可夫状态模型(MSM)，发现PPI的释放经历了一个从空穴跳跃的激活过程，不太可能进一步支持易位。MSM对多亚基RNAP的实施之前证明了PPI释放的电荷促进了跳跃机制。为了进一步探索PPI发布过程中的基本慢动作，我们还进行了微秒长的MD模拟。PPI的释放似乎没有与直接与聚合酶易位相关的O-螺旋的旋转打开紧密耦合。因此，这项研究再次反对权力中风的情景。值得注意的是，我们发现了一个关键残基Lys472，它能够帮助PPI跳出空洞。这种机制可以通用于一组与T7 RNAP具有保守结构特征的聚合酶，而帮助PPI释放的赖氨酸或精氨酸模块可能对单亚基和多亚基RNAP通用，尽管总体结构特征和PPI释放机制明显不同。
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. RNA polymerases (RNAPs) are usually recognized to work as Brownian ratchet machines, with mechanical movements and chemical reactions loosely coupled during transcription elongation. Nevertheless, structural studies on the single-subunit T7 RNAP had suggested an alternative power stroke scenario, which requires the PPi product release reaction tightly couples with or drives the polymerase translocation. To resolve the conflicting views in the mechano-chemical coupling scenario, we conducted atomistic molecular dynamics (MD) simulations to investigate the PPi release mechanism of T7 RNAP. Using a large number of nanosecond short simulations, we constructed the Markov state model (MSM) and found that the PPi release undergoes a jump-from-cavity activation process, unlikely to further support the translocation. The MSM implementations to multi-subunit RNAPs previously demonstrated instead the charge facilitated hopping mechanisms of the PPi release. To further explore essential slow motions during the PPi release, we also performed microsecond long MD simulations. The PPi release does not appear to be tightly coupled to the rotational opening of an O-helix that is directly tied to the polymerase translocation. Hence, the study again disfavors the power stroke scenario. Remarkably, we discovered a key residue Lys472 that is able to assist PPi to jump out of the cavity. The mechanism can be general to a group of polymerases sharing conserved structural features with T7 RNAP, while the lysine or arginine module to assist the PPi release can be universal to both the single and multi-subunit RNAPs, even though the overall structural features and the PPi release mechanisms are significantly different.