Unraveling folding and mechanism of a small model ribozyme

描述（由申请人提供）：核酶是在所有生命领域中发现的大量非蛋白编码rna的理想模型系统，因为它们具有易于检测的生物学功能-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学和生物技术相关性。然而，在它们被发现的四分之一个世纪之后，我们对核酶催化作用的理解与蛋白质酶的催化作用相比仍然相形见绌。在过去的两个资助周期中，PI的小组对我们对小核糖酶类的折叠和机制的理解做出了重大贡献。这一类的所有五个成员都进行了不同程度的研究，特别关注锤头病毒和丁型肝炎病毒（HDV）核酶。作为一个特别有趣的模型系统，发夹核酶也取得了一些重要的发现，我们将在当前的资助期内对其进行跟进，带来我们标志性的生物物理和生化工具的整合。在Specific Aim 1中，我们将检验在单分子水平上观察到的发夹核酶的持续折叠异质性是由特定核苷酸糖的缓慢复制引起的假设。在体外（重新）折叠时，已经观察到许多rna在化学上相同的异构体中存在类似的折叠异质性，但仍然缺乏分子解释。我们最近成功地避免了这种异质性，当从体外转录反应中直接天然纯化RNA时，通过单分子荧光共振能量转移（smFRET），足迹和分子动力学（MD）模拟的组合，为研究发夹核酶折叠异质性的分子基础铺平了道路。在《特定目标2》中，我们将与计算科学家兼长期合作伙伴Jiri Sponer和x射线晶体学家Joseph Wedekind合作，测试发卡核酶中全局分子运动网络对导致催化的局部分子运动有影响的假设。这种联系已被提出用于蛋白质酶，但尚未对任何核酶进行严格的测试。为此，我们将在发夹核酶和探针中引入位点特异性修饰，使用酶学，smFRET， x射线晶体学和MD模拟的组合，研究每种修饰对局部和全局结构，动力学和功能的影响。在Specific Aim 3中，我们将测试一组关于A38和水在发夹核酶催化中的作用的具体机制建议。这个目标是继我们之前的观察，一个明智地放置的A38残留物是由几个紧密结合的水分子在溶剂保护的催化核心的两侧。我们将与Jiri Sponer和Joseph Wedekind以及量子化学家michael Otyepka合作，对催化反应进行广泛取样的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。

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.