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的调控功能中起着至关重要的作用。 许多调节RNA在它们被激活时经历这些功能上重要的构象动力学。 转录，这一过程被称为“共转录折叠”。而内在RNA构象动力学 和共转录折叠可以耦合，以引发生物功能，潜在的机制仍然存在 由于难以表征和检查RNA构象动力学， 共转录折叠的背景。为了实现我们阐明调控RNA功能的长期目标， 该提案的总体目标是整合溶液NMR、计算NMR和核磁共振成像的突破性技术， 建模和时间分辨的化学探测，以揭示通过共转录RNA的调节原理 动力学与核糖开关的具体应用，一类非编码RNA，作为配体- 依赖于基因调节因子，是开发新型抗生素的新兴靶点。在前期融资中 期间，我们已经挑战了核糖开关的传统工作模型，通过显示，在溶液中， 条件下，氟化物核糖开关的传感适体结构域采用相同的构象， 配体的存在或不存在。我们发现，无配体的传感适体经历了明显的 构象动力学涉及低填充和短寿命的激发态（ES），其中ES- 介导的构象转换与共转录折叠协同作用， 依赖性转录激活目前的建议是我们的概念和 技术创新对理解核糖开关功能，我们的目标是建立共同的， 基于转录RNA动力学的调控机制，以推进转录调控的新范式。 和翻译核糖开关，并进行生物化学测定和诱变，以重新设计个体 监管措施，以测试预测。为了实现总体目标，拟议的研究详细介绍了三个方面 具体目标是以逐渐增加的复杂性为特征：（1）表征共转录RNA 转录氟化物核糖开关的动力学，（2）表征转录氟化物核糖开关的共转录RNA动力学。 翻译的氟化物核糖开关，和（3）表征FMN的共转录RNA动力学 核糖开关结果将被用来测试这一提议的核心假设，即RNA结构具有 进化为编码不同的共转录构象动力学，以促进调节结构 沿着沿着特定的功能通路变化。通过深入了解 转录和翻译核糖开关，拟议的研究将阐明的基本性质， 共转录RNA动力学及其在基因调控中的作用。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RNA靶向抗菌剂， 治疗和推进功能RNA的共转录调控的分子理解。