Metal dependent structure, dynamics, and function in RNA measured by site-directed spin labeling and EPR spectroscopy

The structure and function of RNA molecules are dependent on RNA-metal ion interactions in both diffusive and direct ways. Structural information for RNA has been obtained using various biophysical and biochemical methods. In this study, using site-directed spin labeling (SDSL) and EPR spectroscopy, distances in RNA duplexes, TAR RNA, and the hammerhead ribozyme have been measured to investigate RNA structures. Kinetic measurements have been performed in the extended hammerhead ribozyme to correlate the catalytic function with metal dependent ribozyme folding. As a basic model system for distance measurements, inter-spin distances in RNA duplexes with spin labels at various positions are measured using SDSL with continuous EPR and a Fourier deconvolution method. Divalent metal-ion dependent TAR RNA folding from bent to extended conformers is monitored by measuring inter-spin distances near the bulge region. In order to investigate a proposed loop-loop interaction in the extended hammerhead ribozyme which significantly enhances the ribozyme activity, distance measurements, dynamics studies, and kinetics measurements have been performed. We have introduced PELDOR long-distance measurements in order to investigate metal dependent folding of the hammerhead ribozyme. The dynamics of the spin labels attached to the hammerhead ribozyme with increasing mono- and divalent metal ion concentrations are monitored using CW EPR spectroscopy at room temperature. EPR data show that a loop-loop interaction occurs near the U1.6 nucleotide, and that in 0.1 M NaCl the docking occurs at submillimolar Mg2+ concentrations ([Mg2+]1/2, docking = ~ 0.7 mM). Kinetics measurements show that the hammerhead ribozyme requires high concentration of Mg2+ for the maximum cleavage activity ([Mg2+]1/2, cleavage = ~ 90 mM).