Accurate Distance Constraints for RNA Structures Using Deer Spectroscopy

Abstract

Understanding structure-function relationships in RNA and RNA-protein complexes requires robust methods for obtaining structural information on a variety of length scales. DEER (double electron-electron resonance) is emerging as a powerful method for very accurate (± 2 A over 15-80 A) distance measurements between pairs of nitroxide labels that can be placed using several available conjugation sites in RNA nucleobases or phosphodiester linkages. Here, we show the potential for DEER spectroscopy in monitoring global RNA folding and also small changes in RNA structure within a model system that is based on the Hammerhead ribozyme. This catalytically active RNA, a three-helix junction motif with a buried active site, undergoes cation-dependent folding transitions that are linked to activity. Nitroxide labels placed at strategic positions allow helix-docking and active-site core rearrangements to be monitored by measuring the dipolar coupling between paramagnetic sites. This poster will present the results of DEER measurements obtained at both X-band and Q-band, where the higher-frequency Q-band spectroscopy significantly enhances the sensitivity of this technique. Mg2+-dependent global folding, and evidence for a smaller local structural change with higher added metal concentrations, are both observed in this RNA. Since labels can be placed at targeted sites within both nucleic acids and proteins, and there is no inherent limitation on macromolecular size, DEER spectroscopy has potential for obtaining high-resolution structural information in complex RNAs and in large RNA or DNA-protein complexes.