PROJECT SUMMARY RNA folds into structures that perform fundamental cellular functions including gene regulation, the catalysis of essential biochemical reactions, and genome defense. In cells, nascent RNA begins to fold when it emerges from an RNA polymerase during transcription. Consequently, the direction and rate of transcription constrains the structures that RNA can fold into, and nascent RNA molecules can interact directly with transcribing RNA polymerases to control their own synthesis. Predicting how changes in the primary sequence of an RNA will affect its structure and function therefore requires a mechanistic understanding of how RNA folding is coordinated with transcription. Because all RNAs begin to fold cotranscriptionally, understanding how RNA folds into functional secondary and tertiary structures will broadly impact our knowledge of RNA biology, the development of RNA biotechnology, and our ability to identify and address human diseases that are caused by RNA misfolding. However, probing the basic mechanisms of cotranscriptional RNA structure formation remains challenging due to the lack of quantitative methods with sufficient throughput to address the complexity of RNA folding processes. The proposed research will address this challenge by systematically dissecting cotranscriptional RNA folding mechanisms using new tools that can assess how combinatorial sequence perturbations affect RNA structure and function. The initial focus of these studies will be to understand how RNA sequence composition and transcription kinetics coordinate the formation of RNA tertiary structures that enable ligand-mediated transcription regulation by riboswitches. This work will uncover basic RNA folding principles that enable accurate and efficient cotranscriptional RNA structure formation, which will advance our ability to both engineer and characterize RNA systems for biomedical and biotechnological applications. More broadly, these studies will establish a framework for investigating cotranscriptional RNA structure and function that, in the long term, will contribute to a predictive understanding of how RNA folds and how sequence mutations can cause RNA to fold into dysfunctional states.