Riboswitch and Ribozyme Dynamics at Atomic Resolution

 描述(申请人提供)：构象动力学在非编码RNA(NcRNAs)的功能中发挥重要作用，包括核糖开关的基因调控，小分子自裂解核酶的酶催化，以及核糖体的蛋白质合成。构象变化可以在多个步骤中发生，每个步骤都由不同的细胞输入触发，并导致不同的结构，在多步骤生化途径中服务于独特的功能。因此，对ncRNAs的结构、动力学、热力学和动力学的综合认识对于深入理解其生物学机制是至关重要的。核糖开关和核酶是两个经典的ncRNAs，它们在转录和翻译等关键细胞过程中发挥不依赖于蛋白质的调节作用。在这里，我们建议对这两类ncRNAs的动力学机制有一个更深入和全面的了解，以氟化物核糖开关和锤头状核酶为模型系统来举例说明RNA的调节和催化活性。为了阐明核糖开关如何控制基因表达和核酶如何执行催化作用的长期目标，这项拟议研究的总体目标是开发和应用溶液核磁共振方法来可视化控制其机制的自由能景观，并执行生化分析和突变以重新设计单个功能步骤来测试预测。为了实现这一总体目标，这项研究提出了三个具体的目标，其特征是结构和动力学的复杂性逐渐增加：(1)描述氟化核糖开关适配子的配体结合机制，(2)表征适配子与氟化核糖开关表达平台之间的信号转导，以及(3)阐明变构构象动力学在锤头状核酶催化作用中的作用。结果将被用来检验这一提议的中心假设，即RNA结构已经进化到编码复杂的构象景观，以指导沿着特定功能路径的结构变化。这些拟议的研究将有助于更好地从机制上理解核糖开关和核酶的功能，并为研究更复杂的核糖开关和核酶奠定基础。了解核糖开关和核酶的工作原理将进一步帮助开发以核糖开关为靶点的抗微生物疗法、基于核酶的基因敲除工具，以及新的RNA功能的从头设计和精确工程。所提出的高分辨率核磁共振方法也将为该领域提供工具和技术，以促进对其他ncRNA功能的分子理解。

 DESCRIPTION (provided by applicant): Conformational dynamics play essential roles in the functions of non-coding RNAs (ncRNAs), including gene regulation by riboswitches, enzymatic catalysis by small self-cleaving ribozymes, and protein synthesis by ribosomes. The conformational changes can take place in multiple steps, each triggered by distinct cellular inputs and lead to distinct structures that serve unique functions during the multi-step biochemical pathways. Therefore, an integrated structural, dynamic, thermodynamic, and kinetic view of ncRNAs is crucial for developing deep understanding of their biological mechanisms. Riboswitches and ribozymes are two classic ncRNAs that serve as protein-independent regulators in critical cellular processes such as transcription and translation. Here, we propose to develop a deep and comprehensive understanding regarding the dynamic mechanisms of these two classes of ncRNAs, using the fluoride riboswitch and the hammerhead ribozyme as model systems to exemplify RNA's regulatory and catalytic activities. Towards our long-term goal of elucidating how riboswitches control gene expressions and how ribozymes perform catalysis, the overall objective of this proposed research is to develop and apply solution NMR methods to visualize the free energy landscapes that govern their mechanisms and to perform biochemical assays and mutagenesis to reengineer individual functional steps to test predictions. To accomplish this overall objective, the proposed research details three specific objectives that feature a gradual increase in the complexity of structure and dynamics: (1) delineate the mechanism of ligand binding of the fluoride riboswitch aptamer, (2) characterize the signal transduction between the aptamer and the expression platform of the fluoride riboswitch, and (3) elucidate the role of allosteric conformational dynamics in enzymatic catalysis by the hammerhead ribozyme. Results will be used to examine the central hypothesis of this proposal that RNA structures have evolved to encode complex conformational landscapes to direct structural changes along specific functional pathways. These proposed studies will facilitate developing a better mechanistic understanding of riboswitch and ribozyme functions and formulating foundations for studying even more complex riboswitches and ribozymes. Understanding how riboswitches and ribozymes work will further assist the development of riboswitch- targeted antimicrobial therapeutics, ribozyme-based gene knockdown tools, and de novo design and precise engineering of novel RNA functions. The proposed high-resolution NMR methods will also provide the field with tools and techniques for advancing the molecular understanding of other ncRNA functions.