Riboswitch Dynamics at Atomic Resolution

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。 许多调控RNA经历了这些功能上重要的构象动力学，因为它们是 转录，这一过程被称为“共转录折叠”。而固有的RNA构象动力学 而共转录折叠可以偶联来引发生物学功能，其潜在机制仍然存在 由于在描述和研究RNA构象动力学方面的困难，人们对 共转录折叠的上下文。为了我们阐明调控RNA如何发挥作用的长期目标， 这一提议的总体目标是将溶液核磁共振、计算 建模和时间分辨化学探测，以揭示通过共转录RNA进行调控的原理 动力学与核糖开关的具体应用，核糖开关是一类作为配体的非编码RNA- 依赖于基因调节器，是开发新型抗生素的新兴目标。在之前的资助期间 期间，我们对核糖开关的传统工作模式提出了挑战，表明在溶液中 条件下，氟化核糖开关的传感适配子结构域在 配体的存在或不存在。我们发现，无配体的感应适配子经历了不同的 涉及低布居和短寿命激发态(ES)的构象动力学，其中ES- 介导的构象转换与共转录折叠协同工作，调节配体- 依赖转录激活。目前的建议代表了我们的概念和 为了解核糖开关功能而进行的技术创新，我们的目标是建立共同的 基于转录RNA动力学的调控机制，以推进转录的新范式 和翻译核糖开关，并进行生化分析和突变以重新设计个体 检验预测的监管措施。为了实现总体目标，拟议的研究详细介绍了三个方面 以复杂性逐渐增加为特征的特定目标：(1)表征共转录RNA 转录氟化物核糖开关的动力学，(2)表征共转录的RNA动力学 翻译氟核糖开关，以及(3)表征FMN的共转录RNA动力学 核糖开关。结果将被用来检验这一提议的中心假设，即RNA结构具有 进化到编码不同的共转录构象动态，以促进调控结构 沿着特定的功能通路发生变化。通过深入的机械理解 转录和翻译核糖开关，拟议的研究将阐明基本性质 共转录核糖核酸动力学及其在基因调控中的作用。概念框架和实验 建议中开发的工具可以进一步协助RNA靶向抗菌剂的开发 治疗和促进对功能性RNA的共转录调控的分子理解。

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.