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 fur...