Riboswitch Dynamics at Atomic Resolution

原子分辨率下的核糖开关动力学

• 批准号: 10338183
• 负责人: Qi Zhang
• 金额: $ 33.94万
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10338183
• 关键词: 3-Dimensional; Adopted; Anabolism; Antibiotic Resistance; Antibiotics; Bacillus cereus; Bacteria; Binding; Biochemical; Biological Assay; Biological Process; Cells; Chemicals; Code; Communication; Complex; Computer Models; Coupled; Crystallization; Deltaproteobacteria; Development; Flavin Mononucleotide; Fluorides; Funding; Gene Expression Regulation; Genetic Transcription; Goals; Individual; Kinetics; Ligands; Maps; Mediating; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; Pathway interactions; Play; Process; Property; RNA; RNA Conformation; Regulation; Regulator Genes; Research; Resolution; Riboflavin; Ribosomes; Role; Spectrum Analysis; Structural Models; Structure; System; Techniques; Testing; Therapeutic; Thermodynamics; Time; Transcriptional Activation; Transcriptional Regulation; Translational Regulation; Translations; Untranslated RNA; Work; antimicrobial; aptamer; base; conformational conversion; insight; novel; pathogenic bacteria; protein expression; public health relevance; small molecule; synthetic biology; technological innovation; tool

Conformational dynamics play essential roles in the regulatory functions of coding and non-coding RNAs. Many regulatory RNAs undergo these functionally important conformational dynamics as they are being transcribed, a process referred to as ‘co-transcriptional folding’. While intrinsic RNA conformational dynamics and co-transcriptional folding can be coupled to elicit biological functions, the underlying mechanisms remain poorly understood due to difficulties in characterizing and examining RNA conformational dynamics in the context of co-transcriptional folding. Towards our long-term goal of elucidating how regulatory RNAs function, the overall objective of this proposal is to integrate breakthrough techniques of solution NMR, computational modeling, and time-resolved chemical probing to uncover principles of regulation via co-transcriptional RNA dynamics with specific applications to riboswitches, a class of non-coding RNAs that serve as ligand- dependent gene regulators and are emerging targets for developing novel antibiotics. During the prior funding period, we have challenged the conventional working model of riboswitches by showing that, under solution conditions, the sensing aptamer domain of the fluoride riboswitch adopts the same conformation in the presence or absence of the ligand. We found that the ligand-free sensing aptamer undergoes distinct conformational dynamics involving a low-populated and short-lived excited state (ES), where the ES- mediated conformational transition works in coordination with co-transcriptional folding to regulate ligand- dependent transcription activation. The present proposal represents a continuum of our conceptual and technological innovations towards understanding riboswitch functions, in which we aim to establish co- transcriptional RNA dynamics based regulatory mechanisms, to advance new paradigms for transcriptional and translational riboswitches, and to perform biochemical assays and mutagenesis to reengineer individual regulatory steps to test predictions. To accomplish the overall objective, the proposed research details three specific objectives that feature a gradual increase in the complexity: (1) characterize co-transcriptional RNA dynamics of the transcriptional fluoride riboswitch, (2) characterize co-transcriptional RNA dynamics of the translational fluoride riboswitch, and (3) characterize co-transcriptional RNA dynamics of the FMN riboswitches. Results will be used to test the central hypothesis of this proposal that RNA structures have evolved to encode distinct co-transcriptional conformational dynamics to facilitate regulatory structural changes along specific functional pathways. By developing a deep mechanistic understanding of transcriptional and translational riboswitches, the proposed studies will illuminate fundamental properties of co-transcriptional RNA dynamics and its role in gene regulation. The conceptual framework and experimental tools developed in the proposal can further assist the development of RNA-targeted antimicrobial therapeutics and advance the molecular understanding of co-transcriptional regulation of functional RNAs.

构象动力学在编码和非编码RNA的调节功能中起着至关重要的作用。 许多调节性RNA在其上都经历了这些功能上重要的构象动力学 转录，一个称为“共同折叠”的过程。而固有的RNA构象动力学 并可以将共转录折叠耦合到引起生物学功能，潜在的机制仍然存在 由于难以表征和检查RNA构象动态，因此理解不足 共同折叠的上下文。达到我们阐明监管RNA功能的长期目标， 该提案的总体目的是整合解决方案NMR的突破性技术 建模和时间分辨的化学探测以发现通过共转录RNA的调节原理 具有特定应用到核糖开关的动力学，这是一类非编码RNA，用作配体 依赖基因调节剂，是开发新型抗生素的新兴靶标。在先前的资金期间 时期，我们通过证明在解决方案下向核糖开关的常规工作模型提出了挑战 条件，氟化物核糖开关的感应蚀面域适应了相同的构象 配体的存在或不存在。我们发现，不含配体的感觉aper被不同 构象动力学涉及一个低人口且短暂的激发态（ES），其中ES- 介导的会议过渡与共同折叠的协调作用，以调节配体 依赖转录激活。本提案代表了我们的概念和 了解Riboswitch功能的技术创新，我们旨在建立共同 基于转录RNA动力学的调节机制，以推动转录的新范式 并翻译了核糖开关，并进行生化测定和诱变为重新设计师个体 测试预测的监管步骤。为了实现总体目标，拟议的研究详细介绍了三 具有复杂性等级的特定目标：（1）表征共转录RNA 转录氟化物核糖开关的动力学，（2）表征了共转录RNA动力学 翻译氟化物核糖开关，（3）表征FMN的共转录RNA动力学 核糖开关。结果将用于检验该提案的中心假设，即RNA结构已有 演变为编码不同的共转录构象动力学以促进调节结构 沿特定功能途径的变化。通过对 转录和翻译核糖开关，拟议的研究将阐明 共转录RNA动力学及其在基因调节中的作用。概念框架和实验 该提案中开发的工具可以进一步帮助开发以RNA为目标的抗菌剂 疗法并提高对功能RNA共转录调控的分子理解。