Development of NMR methodology for probing functional dynamics in RNA.

Abstract

Over last two decades, several new RNA functions have been discovered completely transforming how we view its role in gene expression and regulation. A key feature of many of these new RNA functions is that they almost always require or involve dramatic conformational changes in the RNA that typically occur in response to protein or ligand recognition. Though it is clear that RNA structures must change to perform their functions, the mechanism by which this occurs remains poorly understood. A central problem is that the dynamical properties of nucleic acids remain poorly understood especially in comparison to proteins. This dissertation describes the development of solution nuclear magnetic resonance (NMR) spectroscopic methods for characterizing dynamics in nucleic acids at timescales ranging between picoseconds and milliseconds. The methods are applied to uncover the molecular basis by which the transactivation response element (TAR) RNA from the human immunodeficiency virus type 1 (HIV-1) genome adaptively changes its structure and thereby binds to dramatically different molecular targets. A major problem in the application of NMR towards the study of RNA dynamics is that internal motions can lead to correlated changes in overall motions making analysis of NMR parameters intractable. This limitation is addressed by analytical methods for the case of magnetic field induced residual dipolar couplings (RDCs) and by chemical elongation of a target helix in the case of ordering media induced RDCs and spin relaxation measurements. The combined application of RDCs and spin relaxation allowed the characterization of the entire dynamical spectrum of TAR from picosecond to millisecond timescales. Results show that the two helices in TAR undergo super large amplitude motions at suprananosecond timescales. The helices twist and bend in a highly correlated manner and allow free TAR to dynamically visit seven distinct ligand bound conformation. The helix motions are activated by organized local motions in the bulge linker and neighboring residues where small molecules and ligands bind. Our results strongly suggest that small molecules capture existing TAR conformations that are appreciably populated in the free state rather than induce new ones via the conventional induced-fit mechanism.