Riboswitch and Ribozyme Dynamics at Atomic Resolution

原子分辨率下的核糖开关和核酶动力学

• 批准号: 9061748
• 负责人: Qi Zhang
• 金额: $ 28.59万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9061748
• 关键词: Affect; Bacillus cereus; Bacteria; Binding; Biochemical; Biochemical Pathway; Biological; Biological Assay; Biological Models; Catalysis; Catalytic Domain; Catalytic RNA; Cell physiology; Chemicals; Cleaved cell; Communication; Complex; Development; Docking; Engineering; Ensure; Equilibrium; Fluoride Ion; Fluorides; Foundations; Free Energy; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Geometry; Goals; Health; Human; Individual; Kinetics; Knowledge; Lead; Life; Ligand Binding; Ligands; Magnesium; Methods; Molecular; Molecular Conformation; Mutagenesis; Mutation; Pathway interactions; Play; Process; Property; Protein Biosynthesis; Proteins; RNA; Research; Resolution; Ribosomes; Role; Signal Transduction; Structure; System; Techniques; Testing; Therapeutic; Thermodynamics; Time; Translations; Untranslated RNA; Work; antimicrobial; aptamer; base; conformational conversion; conformer; design; gene therapy; hammerhead ribozyme; insight; knock-down; meetings; novel; pathogenic bacteria; temporal measurement; tool

 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.

 描述（由申请人提供）：构象动力学在非编码 RNA (ncRNA) 的功能中发挥着重要作用，包括核糖开关的基因调控、小型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，并导致在多步骤生化途径中发挥独特功能的不同结构。因此，对 ncRNA 的结构、动力学、热力学和动力学的综合看法对于深入了解其生物学机制至关重要。核糖开关和核酶是两种经典的 ncRNA，在转录和翻译等关键细胞过程中充当蛋白质独立的调节因子。在这里，我们建议对这两类 ncRNA 的动态机制有深入而全面的了解，使用氟化物核糖开关和锤头核酶作为模型系统来例证 RNA 的调节和催化活性。为了实现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，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，并进行生化测定和诱变以重新设计各个功能步骤来测试预测。为了实现这一总体目标，该研究详细介绍了三个具体目标，其特征是结构和动力学的复杂性逐渐增加：（1）描绘氟化物核糖开关适体的配体结合机制，（2）表征适体与氟化物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酶催化中的作用 由锤头核酶。结果将用于检验该提案的中心假设，即 RNA 结构已经进化为编码复杂的构象景观，以指导沿着特定功能途径的结构变化。这些拟议的研究将有助于更好地理解核糖开关和核酶功能的机制，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有助于核糖开关靶向抗菌疗法、基于核酶的基因敲除工具以及新型 RNA 功能的从头设计和精确工程的开发。所提出的高分辨率 NMR 方法还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。