【作者】 张蕾；

【导师】 李春华；

【作者基本信息】 北京工业大学 ， 生物医学工程（专业学位）， 2015， 硕士

【摘要】 核糖开关是非编码RNA的一种,通过应答于特定的代谢物结合发生结构的变化来调控基因表达。因此,阐明和描述核糖开关的折叠和去折叠过程对于理解它们的调控机理非常重要。本文中我们首先通过使用基于拓扑的高斯网络模型探索了add型腺嘌呤核糖开关的去折叠过程。模型通过模拟碱基间天然接触的打断来模拟核糖开关的去折叠过程,打断顺序的依据是碱基间的距离涨落。模拟得到了完整的去折叠事件序列,并且此序列与单分子力显微镜的实验结果相符。由于模拟中仅考虑了碱基间的天然接触,这样的结果表明在add型腺嘌呤核糖开关的去折叠过程中其天然拓扑结果起了很大作用。另外,本文中也使用拉伸分子动力学模拟的方法模拟了腺嘌呤核糖开关的去折叠路径。分子动力学模拟是获得生物分子微观运动过程的重要工具,广泛运用于生物分子的动力学研究。然而直接模拟生物分子的完整去折叠路径由于所需的计算时间过长,受限于目前计算机的硬件水平,存在一定的困难。而受控分子动力学中的拉伸分子动力学,通过对分子施加外力的方法来加快去折叠的过程,缩短了模拟时间,因此可以把计算时间控制在可接受的范围内。本论文包含两部分工作:(1)利用高斯网络模型研究腺嘌呤核糖开关的去折叠路径。利用课题组在研究蛋白质去折叠路径过程中建立的迭代高斯网络模型的方法,对腺嘌呤核糖开关的去折叠过程进行了模拟和分析,同时也考察了天然构象下金属离子和配体对结构的影响。迭代模拟的结果与实验获得的去折叠路径能较好的吻合。对于模拟结果,我们分析了碱基间涨落的交叉相关性随去折叠过程的变化,交叉相关性的变化显示非天然态似乎有整体共同运动的趋势。同时我们也分析了快运动模式的涨落随去折叠过程的变化,结果显示靠近结合口袋的区域涨落幅度更大,且这部分的涨落一直保留到去折叠过程的后期。这从侧面说明这些靠近结合口袋的区域去折叠的时间较晚。最后分析了镁离子和配体对核糖开关动力学特性的影响,结果表明,在天然态的add型腺嘌呤核糖开关中镁离子和配体显示出一定程度的预组织和稳定结构的作用;对残基涨落的分析表明它们使核糖开关的折叠态结构更加稳定。(2)用拉伸分子动力学方法模拟腺嘌呤核糖开关的去折叠过程。为了在有限的模拟时间内获得完整的去折叠路径,拉伸分子动力学的方法被用来模拟核糖开关的去折叠。得出的二级结构的去折叠顺序与实验结果基本相符。此外我们也分析了loop区和连接区的原子间相互作用情况随时间的变化曲线,结果表明在未结合配体的状态下连接区部分的堆叠作用有所减弱。更多还原

【Abstract】 Riboswitches are noncoding mRNA elements that can regulate the gene expression via altering their structures in response to specific metabolite binding. Therefore, elucidating and characterizing the folding/unfolding process of riboswitches are very important for understanding their regulatory mechanisms.In this dissertation we studied the unfolding behavior of add A-riboswitch through the iterative topology-based Gaussian network model. In this model the unfolding behavior of add A-riboswitch was mimicked by breaking the native contacts between different nucleotides according to the fluctuations of the distance between them. The complete sequence of add A-riboswitch unfolding event was obtained, which is consistent well with the experimental data of single molecule force measurements. Since only the native contacts are considered during the simulation, this result suggests that the native topology may play an important role in the add A- riboswitch unfolding process.Additionally, steered molecular dynamics simulation was used to explore the unfolding pathway of add A-riboswitch, too. Molecular dynamics simulation is an important method to investigate the detailed microscopic dynamics of biomolecules and has been widely used in the field of dynamic studies of biological macromolecules. However, due to the expensive computational costs, it is hard to simulate the whole unfolding process. Instead we used steered molecular dynamics simulation to explore the unfolding behavior, which was speeded up largely by external force; therefore the simulation can be done in acceptable time scale.There are two contents in this thesis:(1) Explore the unfolding pathway of add A-riboswitch with the iterative Gaussian network model. Using the iterative Gaussian network model proposed by our group in studying protein unfolding pathway, we simulated the add A-riboswitch’s unfolding behavior and analyzed metal ion and ligand’s effects onthe native conformation. Instead of using P atoms as nodes as in conventional Gaussian network model method, we chose the atomsof bases to mark different types of nucleotides. These mark atoms was chosen based on Watson-Crick base-pairing. The results of iterative simulations show a complete unfolding pathway which is consistent with the experimental data. After that we analyzed the change of cross-correlations between nucleotide fluctuations during the unfolding process, and the results indicate the denatured state seems to move highly cooperatively. In addition, we investigated fluctuations’ change in the fast modes during unfolding process. It’s found that regions close to binding pocket hold higher fluctuations. At last we analyzed the effects of Mg2+ ions and the ligand on the dynamical properties of add A-riboswitch. The results display that they are helpful in pre-organization in some degree, as well as helpful in stabilizing the folded conformation of the riboswitch.(2) Study the unfolding process of add A-riboswitch by steered molecular dynamics simulations. In order to obtain the complete unfolding process in acceptable time scale,the external force was put on the riboswitch molecular. The unfolding pathway we got with steered molecular dynamics simulation is roughly consistent with experimental results. In additional we analyzed the changes of interactions between atoms in loop-loop region and junctions region. The result indicate that the base stacking interactions is reduced when the ligand gone.更多还原