Unraveling folding and mechanism of a small model ribozyme

描述(申请人提供)：核酶是生命所有领域中发现的大量非蛋白质编码RNA的理想模型系统，因为它们具有易于检测的生物功能-催化。由于它们在处理和管理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，在他们发现四分之一个世纪后，我们对核酶催化的理解仍然比不上蛋白质酶的催化。在过去的两个资助周期中，PI的小组为我们理解小核酶的折叠和机制做出了实质性的贡献。这一类别的所有五名成员都进行了不同程度的调查，特别是锤头和丁型肝炎病毒(HDV)核酶。发夹核酶作为一种特别耐人寻味的模型系统也有几项重要发现，我们将在当前的资助期内对其进行跟踪，使我们标志性的生物物理和生化工具集成在一起。在特定的目标1中，我们将检验这样的假设，即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再折叠引起的。在体外折叠时，许多RNA在化学上相同的异构体也观察到了相似的折叠异质性，但仍然缺乏分子解释。我们最近成功地从体外转录反应中直接从天然纯化的RNA中避免了这种异质性，为通过单分子荧光共振能量转移(SmFRET)、足迹和分子动力学(MD)模拟研究发夹状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者Jiri Sponer和X射线结晶学家Joseph Wedekin合作，测试发夹核酶中的全球分子运动网络对导致催化的局部分子运动的影响这一假设。蛋白质酶存在这样的关联，但还没有对任何核酶进行严格的测试。为此，我们将把位点特异性的修饰引入发夹状核酶中，并结合酶学、smFRET、X射线结晶学和MD模拟，探索每一种修饰对局部和整体结构、动力学和功能的影响。在特定的目标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特定机制建议。这一目标延续了我们之前的观察，即在溶剂保护的催化核心中，A38残基被几个紧密结合的水分子包围。我们将与Jiri Sponer和Joseph Wedekin以及量子化学家Michal Otyepka合作，对催化反应进行广泛采样的QM/MM处理。我们预计，这三个特定目标的结果将极大地加深我们对非编码RNA一般生物学功能的理解。

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.