Unraveling folding and mechanism of a small model ribozyme

描述（由申请人提供）：核酶是在生命的所有领域中发现的大量非蛋白质编码RNA的理想模型系统，因为它们具有容易检测的生物学功能-催化。由于它们在处理和调节遗传信息方面的作用，它们本身也具有高度的生物和生物技术相关性。然而，在它们被发现后的四分之世纪，我们对核酶催化作用的理解与蛋白酶催化作用相比仍然相形见绌。在过去的两个资助周期中，PI的团队为我们理解这类小核酶的折叠和机制做出了重大贡献。该类的所有五个成员都进行了不同程度的研究，特别关注锤头和丁型肝炎病毒（HDV）核酶。发夹状核酶作为一个特别有趣的模型系统也有几个重要的发现，我们将在当前的资助期内跟进，将我们标志性的生物物理和生物化学工具整合起来。在特定目标1中，我们将测试以下假设：在单分子水平上观察到的发夹状核酶的持续折叠异质性是由特定核苷酸糖的缓慢重新折叠引起的。当在体外折叠（再）时，已经观察到许多RNA的化学相同的异构体的类似折叠异质性，但仍然缺乏分子解释。我们最近已经成功地避免了这种异质性时，天然纯化的RNA直接从体外转录反应，铺平了道路，调查折叠异质性的发夹状核酶的分子基础相结合的单分子荧光共振能量转移（smFRET），足迹，和分子动力学（MD）模拟。在《特定目标2》中，我们将与计算科学家和长期合作者Jiri Sponer以及X射线晶体学家Joseph Wedekind合作，测试发夹状核酶中的全局分子运动网络对导致催化的局部分子运动产生影响的假设。这种连接已被建议用于蛋白酶，但尚未对任何核酶进行严格的测试。为此，我们将引入位点特异性修饰的发夹核酶和探针，使用酶学，smFRET，X射线晶体学和MD模拟的组合，这些修饰的局部和全局的结构，动力学和功能的影响。在具体目标3中，我们将测试A38和水在发夹核酶催化中的作用的一组具体机制建议。这一目标是根据我们先前的观察，即明智地放置的A38残基在溶剂保护的催化核心中被几个紧密结合的水分子侧接。我们将与Jiri Sponer和Joseph Wedekind以及量子化学家Michal Otyepka合作，对催化反应进行广泛的QM/MM处理。我们预计，这三个特定目标的结果将大大加深我们对非编码RNA生物学功能的理解。 公共卫生相关性：核酶是在生命的所有领域中发现的大量非蛋白质编码RNA的理想模型系统，因为它们具有容易检测的生物学功能-催化。由于它们在处理和调节遗传信息方面的作用，它们本身也具有高度的生物和生物技术相关性。在这个项目的更新中，一个小的模型核酶，发夹核酶，三个神秘的标志，将机械解剖，以加深我们对生物学相关的非编码RNA的一般理解。

DESCRIPTION (provided by applicant): Ribozymes are ideal model systems for the vast number of non-protein coding RNAs found in all domains of life, since they have an easily detectable biological function - catalysis. They also are of high biological and biotechnological relevance in their own right for their roles in the processing and regulation of genetic information. Yet, a quarter century after their discovery, our understanding of catalysis by ribozymes still pales compared to that of catalysis by protein enzymes. Over the last two funding cycles, the PI's group has made substantial contributions to our understanding of the folding and mechanism of the class of small ribozymes. All five members of this class were investigated to varying degrees, with particular focus on the hammerhead and hepatitis delta virus (HDV) ribozymes. Several important discoveries were also made on the hairpin ribozyme as a particularly intriguing model system, on which we will follow up during the current funding period, bringing to bear our signature integration of biophysical and biochemical tools. In Specific Aim 1, we will test the hypothesis that the persistent folding heterogeneity of the hairpin ribozyme, observed at the single molecule level, is caused by slow repuckering of specific nucleotide sugars. Similar folding heterogeneity of chemically identical isomers has been observed for a number of RNAs when (re)folded in vitro, but still lacks a molecular explanation. We have recently succeeded in avoiding this heterogeneity when natively purifying the RNA directly from an in vitro transcription reaction, paving the way for investigating the molecular basis of folding heterogeneity in the hairpin ribozyme by a combination of single molecule fluorescence resonance energy transfer (smFRET), footprinting, and molecular dynamics (MD) simulations. In Specific Aim 2, in collaboration with Jiri Sponer, a computational scientist and long-standing collaborator, and Joseph Wedekind, an X-ray crystallographer, we will test the hypothesis that a network of global molecular motions in the hairpin ribozyme has an impact on those local molecular motions that lead to catalysis. Such a linkage has been suggested for protein enzymes, but has not been rigorously tested for any ribozyme. To this end, we will introduce site-specific modifications into the hairpin ribozyme and probe, using a combination of enzymology, smFRET, X-ray crystallography, and MD simulation, the impact of each of these modifications on local and global structure, dynamics, and function. In Specific Aim 3, we will test a set of specific mechanistic proposals for the role of A38 and water in catalysis of the hairpin ribozyme. This aim follows up on our previous observation that a judiciously placed A38 residue is flanked in the solvent- protected catalytic core by several tightly bound water molecules. We will pursue a broadly sampled QM/MM treatment of the catalytic reaction in collaboration with Jiri Sponer and Joseph Wedekind, as well as quantum chemist Michal Otyepka. We anticipate that results from these three Specific Aims will significantly deepen our understanding of the biological function of non-coding RNAs in general. PUBLIC HEALTH RELEVANCE: Ribozymes are ideal model systems for the vast number of non-protein coding RNAs found in all domains of life, since they have an easily detectable biological function - catalysis. They also are of high biological and biotechnological relevance in their own right for their roles in the processing and regulation of genetic information. In this project renewal, three enigmatic hallmarks of a small model ribozyme, the hairpin ribozyme, will be mechanistically dissected to deepen our understanding of biologically relevant non-coding RNAs in general.