分子动力学方法研究RNA聚合酶的移位和选择机理

发布时间：2019-04-12 16:18

【摘要】：RNA聚合酶(RNAP)在遗传信息的表达过程当中起至关重要的作用:在DNA链上移动并将包含遗传信息的DNA链转录为RNA链。RNA聚合酶包含单体和多体两种。在转录过程中,最重要的两个步骤是:RNAP沿DNA链的移位(translocation)和RNAP对底物核糖核苷酸的选择(selection)。RNAP一次可以转录上百到上千个核苷酸,在这个持续过程中RNAP的机械移动机制一直是研究的热点和重点。遗传信息的保真度对生命体而言至关重要,在转录过程中与保真度相关的就是RNAP对底物核苷酸的选择机制。近几十年的研究已经对RNAP的工作机制提供了很多信息,但仍然有很多地方还不清楚。分子动力学(Molecular Dynamics,MD)模拟作为实验的重要补充在近些年的研究中扮演了不可替代的角色。本文利用分子动力学模拟的方法对RNA聚合酶的移位机制和选择机制进行了研究,为其工作机制的研究提供了重要的信息。全文主要包括以下几个部分:多体RNA聚合酶Pol II的移位机制研究。通过和细菌内的多体RNAP结构的对比,一个螺旋(Bridge Helix,BH)的flipped-out构象和straight构象之间的转变被赋予了重要的意义。然而,结晶结构并未在Pol II中发现BH的flipped-out构象。通过分子动力学的模拟,我们发现了2个BH的flipped-out构象,这两个构象对RNAP的移位过程均有促进作用,而且其中一个结构与结晶结构在细菌中发现的BH的flipped-out构象非常类似,证明了BH的flipped-out构象在Pol II中的存在。单体T7 RNA聚合酶的移位机制研究。T7 RNAP作为研究单体RNAP的模型,关于其移位机理主要有:Power-Stroke模型和布朗棘轮(Brownian-Ratchet)模型。我们分子动力学模拟的结果表明:T7 RNAP的移位机理为布朗棘轮模型。我们进一步解释了实验中移位前后的能量差的来源。计算结果与实验结果定量吻合。单体T7 RNA聚合酶的选择机制研究。我们通过分子动力学模拟发现,在T7 RNAP移位过程中存在一个中间态。在这个中间态中,将要作为模板配对的过渡DNA核苷酸(TN)能够移位到活性位点,参与对底物NTP的选择。这个结构并没有相应的结晶结构。我们利用分子动力学模拟对这个状态时NTP的选择机制进行了研究。我们发现,NTP的选择可以依靠NTP与DNA模板碱基之间的WC(Watson-Crick)相互作用,活性位点附近的稳定性和Y639残基。这可能揭示了传统选择路径之外的另一条选择路径。
[Abstract]:RNA polymerase (RNAP) plays an important role in the expression of genetic information: it moves through the DNA chain and transcribes the DNA chain containing genetic information into the RNA chain. RNA polymerase contains two kinds of monomers and polysomes. Two of the most important steps in the transcription process are the transfer of RNAP along the DNA strand (translocation) and RNAP's choice of substrate ribonucleotides (selection). RNAP can transcribe hundreds to thousands of nucleotides at a time. In this continuous process, the mechanism of mechanical movement of RNAP has always been the focus and focus of research. The fidelity of genetic information is very important to the whole life. In the process of transcription, the selection mechanism of substrate nucleotides by RNAP is related to fidelity. The research of recent decades has provided a lot of information about the working mechanism of RNAP, but there are still many things that are not clear. Molecular dynamics (Molecular Dynamics,MD) simulation plays an irreplaceable role in recent studies as an important complement to experiments. In this paper, the molecular dynamics simulation method was used to study the mechanism of RNA polymerase translocation and selection, which provided important information for the study of its working mechanism. The main contents of this paper are as follows: studies on the translocation mechanism of multi-body RNA polymerase Pol II. By comparing with the multi-body RNAP structure in bacteria, the transition between the flipped-out conformation and the straight conformation of a Bridge Helix,BH is of great significance. However, the flipped-out conformation of BH was not found in Pol II. By molecular dynamics simulation, we found two flipped-out conformations of BH, both of which promote the translocation of RNAP, and one of them is very similar to the flipped-out conformation of BH found in bacteria with crystalline structure. The existence of flipped-out conformation of BH in Pol II was proved. The translocation mechanism of monomer T7 RNA polymerase was studied. As a model for studying monomer RNAP, T7 RNAP was used as the model of monomeric RNAP. The shift mechanism of T7 PCR was mainly Power-Stroke model and Brownian ratchet (Brownian-Ratchet) model. The results of our molecular dynamics simulation show that the shift mechanism of T 7 RNAP is Brownian ratchet model. We further explain the source of the energy difference before and after the shift in the experiment. The calculated results are in good agreement with the experimental results. Selection mechanism of monomer T7 RNA polymerase. We found that there is an intermediate state in the process of T7 RNAP shift by molecular dynamics simulation. In this intermediate state, the transition DNA nucleotide (TN), which will be paired as a template, can be transferred to the active site to participate in the selection of substrate NTP. This structure has no corresponding crystalline structure. We use molecular dynamics simulation to study the selection mechanism of NTP in this state. We found that the selection of NTP can rely on the WC (Watson-Crick) interaction between NTP and DNA template bases, the stability near the active site and the Y639 residue. This may reveal another alternative to the traditional selection path.
【学位授予单位】：中国科学院大学(中国科学院物理研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q75

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