Pathways to Tertiary Structure Interfaces in RNA: An Integrated NMR, Biochemical, and Computational Approach

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RNA, a close chemical cousin of DNA, is most familiar in its messenger role of conveying information from the DNA genome to the cellular machinery that synthesizes proteins encoded by that genome. In recent decades, however, a great variety of noncoding roles for RNA molecules have been discovered, including in catalyzing chemical reactions, regulating gene expression, and in specialized functions such as CRISPR. To fulfill these roles, RNA takes up a wide variety of specific three-dimensional shapes and forms. Relatively little is known, however, about the formation of these three-dimensional RNA structures, leaving large gaps in the search for a predictive framework for the design of functional RNA molecules and of other molecules that target cellular RNAs. This project will carry out an integrated program of spectroscopic, biophysical, biochemical, and computational analyses in order to map out the energetics and folding pathways for RNA structures. The work will provide a full atomic-level description of the formation of an RNA three-dimensional structural interaction, and a scientific understanding of such RNA-RNA interactions in noncoding systems in general. This proposal will train underrepresented groups at the graduate and undergraduate levels. The PI’s efforts in mental-health awareness in the scientific workforce at regional and national conferences will be expanded.Although the factors underlying secondary structure (helix formation) in DNA and RNA are relatively well understood, the detailed study of the driving forces and mechanisms of formation of complex RNA tertiary structures has not kept pace with the great recent advances in RNA structural biology. The docking transition in the hairpin ribozyme is a rare example of a tertiary-only interaction amenable to intermolecular biophysical and spectroscopic investigation, with a wealth of existing structural and functional data and multiple functional readouts. This project will use a combination of NMR spectroscopy and molecular dynamics computations to examine conformational sampling of the individual loops and use functional assays of dynamically-quenched variants to determine the relevance of the observed motional modes determined in each loop and derive internally-consistent models of docking-competent states. An innovative protocol of NMR and Activity-initialized Iterative Markov Analysis (NAIMA) will be used to integrate all of the data in a computational determination of the pathway for the overall docking process. In the course of this work, the derivation of a reference database of unfolded RNA NMR chemical shifts for the general use of the field will be completed. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.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.