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（应用程序提供）：构象动力学在非编码RNA（NCRNA）功能中起着重要作用，包括核糖开关调节基因，小型自我裂解核酶的酶促催化以及核糖体合成蛋白质。组成变化可以以多个步骤进行，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生物化学途径中起着独特的功能。因此，NCRNA的综合结构，动态，热力学和动力学视图对于发展其生物学机制至关重要。核糖开关和核酶是两种经典的NCRNA，在关键细胞过程（例如转录和翻译）中用作蛋白质独立的调节剂。在这里，我们建议使用Fluoride Riboswitch和Hammerhead Ribozyme作为模型系统，以对这两类NCRNA的动态机制有深入而全面的了解，以免除RNA的调节和催化活性。为了我们阐明核能如何控制基因表达以及核酶进行催化的长期目标，这项拟议的研究的总体目标是开发和应用解决方案NMR方法，以可视化控制其机制并进行生物化学测定和进行杂种的自由能景观，以重新启动个人功能进行预测。 To accomplish this overall objective, the proposed research details three specific objectives that feature a grade increase in the complexity of structure and dynamics: (1) delineate the mechanism of ligand binding of the fluoride riboswitch, (2) characterize the signal transduction between the apater and the expression platform of the fluoride riboswitch, and (3) elucidate the role of allosteric conformational Dynamics in enzymatic catalysis by the hammerhead核酶。结果将用于检查该提案的中心假设，即RNA结构已演变为编码复杂的构象景观，以直接沿特定功能途径的结构变化。这些提出的研究将对核糖开关和核酶功能的机械理解以及为研究更复杂的核糖开关和核酶的制定基础提供更好的机械理解。了解核糖开关和核酶的工作将进一步有助于开发核糖开关靶向抗微生物疗法，基于核酶的基因敲低工具以及从头设计和新型RNA功能的精确工程。提出的高分辨率NMR方法还将为该领域提供工具和技术，以推进对其他NCRNA功能的分子理解。