Exploring the thermodynamics of the diversity of divalent magnesium ion interactions with native, partially folded, and unfolded RNA structures

Because RNA is a polyelectrolyte, folding an RNA into a more compact structure creates an electrostatic problem: the unfavorable repulsion of negatively-charged phosphate groups that are brought close together in tertiary contacts. Consequently, the RNA folding reaction is very sensitive to the types and concentrations of ions present in solution, especially the divalent Mg2+ ion, which is a potent stabilizer of RNA tertiary structure. Mg2+ ions can interact with RNA in different ways to moderate the electrostatic repulsion between neighboring phosphate groups. The importance of diffuse (fully hydrated) ions in stabilizing RNA structures has been established, but the relative contribution of site-bound (and partially dehydrated) ions remains unknown. Although applications of binding formalisms to ion-RNA interactions currently predominate the literature, a general formalism, based on interaction coefficients, more accurately describes the contributions of ions interacting from different environments. This model-independent formalism has been used to interpret the experiments presented here.;The thermodynamic data for several RNAs stabilized by ions using different modes of interaction are compared: an adenine-binding riboswitch that is primarily stabilized by diffuse ions, and the extremely compact structures of the Mg 2+-binding riboswitch and a 58-nucleotide ribosomal RNA fragment that each bind ions at specific sites in crystal structures and exhibit a high degree of phosphate burial. A fluorescence-based method is employed to measure the excess Mg2+ (Gamma2+) associating with native and partially unfolded forms of these RNAs. Combined with global (from small-angle X-ray scattering) and local (from hydroxyl radical footprinting) structural information, these measurements provide a comprehensive look at how ions stabilize RNA structures and unveil some general principles of RNA folding: (i) a partially unfolded RNA samples native tertiary contacts, (ii) chelated ions allow RNA phosphate backbones to explore regions of conformational space otherwise electrostatically inaccessible, and (iii) the properties of the unfolded RNA are important in determining ion uptake in a folding reaction.