Riboswitch and Ribozyme Dynamics at Atomic Resolution

 描述（由申请人提供）：构象动力学在非编码RNA（ncRNA）的功能中起重要作用，包括核糖开关的基因调控、小自切割核酶的酶催化和核糖体的蛋白质合成。构象变化可以在多个步骤中发生，每个步骤由不同的细胞输入触发，并导致在多步生化途径中发挥独特功能的不同结构。因此，一个完整的结构，动力学，热力学和动力学的观点ncRNA是至关重要的发展深入了解其生物学机制。核糖开关和核酶是两种典型的ncRNA，在关键的细胞过程如转录和翻译中充当蛋白质非依赖性调节剂。在这里，我们建议发展一个深入和全面的了解这两类ncRNA的动态机制，使用氟化物核糖开关和锤头状核酶作为模型系统，以抑制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以及核酶如何进行催化的长期目标，本研究的总体目标是开发和应用溶液NMR方法来可视化管理其机制的自由能景观，并进行生化测定和诱变，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，拟议的研究详细介绍了三个具体目标，这些目标的特点是结构和动力学的复杂性逐渐增加：（1）阐明氟核糖开关适体的配体结合机制;（2）表征适体与氟核糖开关表达平台之间的信号转导;（3）阐明变构构象动力学在锤头状核酶酶促反应中的作用。结果将被用来检查这一建议的核心假设，RNA结构已演变为编码复杂的构象景观，直接结构变化沿着特定的功能途径。这些拟议的研究将有助于开发一个更好的机制理解核糖开关和核酶的功能，并制定研究更复杂的核糖开关和核酶的基础。理解核糖开关和核酶如何工作将进一步有助于核糖开关靶向的抗微生物治疗剂、基于核酶的基因敲除工具以及新RNA功能的从头设计和精确工程的开发。提出的高分辨率NMR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.