Collaborative Research: Uncovering How Riboswitches Exploit Out-of-Equilibrium RNA Folding Pathways to Make Genetic Decisions

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This work will uncover new principles by which riboswitch RNAs exploit out-of-equilibrium RNA folding and ligand binding to make genetic decisions. The tools developed will also be applicable to understanding the role of co-transcriptional folding and assembly of ribozymes, regulatory RNA structures within non-coding and coding RNAs, and assembly of RNA-protein complexes such as the ribosome and spliceosome. These studies will also contribute understanding of the physical principles of out-of- equilibrium co-transcriptional RNA folding, help address long-standing questions about how dynamic RNA structures coordinate genetic processes, and shed light on how natural RNAs exploit out-of- equilibrium mechanisms to efficiently fold on extremely rugged free-energy landscapes. Since co-transcriptional RNA folding happens every time an RNA is synthesized, the broader impacts of this research include developing general principles and techniques that can be used to understand a wide array of fundamental cellular processes from gene expression to regulation. The study of riboswitches also has several broader impacts towards societal goals, since they can be used as biosensors within new molecular diagnostics, and they are important targets for new classes of antibiotics. Broader impacts of integrated research and education will come from a multi-pronged plan including conducting demonstrations of riboswitch diagnostics to school-age groups, mentorship of undergraduate researchers, and delivering hands-on tutorials of computational RNA folding approaches to broader scientific communities.The overarching goals of this proposal are to: (i) Uncover detailed mechanisms of how ligand binding bifurcates out-of-equilibrium RNA cotranscriptional folding pathways to enact genetic decisions in riboswitch RNAs; and (ii) Develop and apply new hybrid experimental-computational frameworks that can reconstruct RNA cotranscriptional folding pathways at the secondary and tertiary structure levels. The education plan focuses on integrating this research into hands-on demonstration activities targeted towards school age children, undergraduate researcher mentorship, and hands-on training tutorials for the broader scientific community. The post-genomic era has ushered in a new appreciation that RNAs play central roles in regulating, maintaining and defending the genomes of all organisms. However, a critical knowledge gap remains: we have relatively little understanding of the dynamic folding pathways that RNAs undergo as they are being synthesized during transcription, thus hindering our fundamental understanding of how RNA structures enact critical cellular functions such as catalysis, gene expression regulation, and cellular sensing. To address this gap, the PIs recently innovated and validated a hybrid experimental-computational approach that uses high-throughput RNA structure chemical probing data with computational algorithms to generate two and three-dimensional models of RNA cotranscriptional folding pathways. One central objective of this proposal is to extend this approach to incorporate more complex RNA structures and interactions such as pseudoknots relevant to a broad range of functional cellular RNAs. The second is to uncover biophysical principles of how out-of-equilibrium RNA fluctuations during cotranscriptional folding influence RNA function. The latter will be pursued through the use of riboswitch RNAs as model systems, which make ligand-mediated genetic decisions, use the dynamic formation of broadly utilized RNA structures to do so, and have broader impact relevance for fundamental biology and biotechnologies.This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.