Evidence for a Thermodynamically Distinct Mg2+ Ion Associated with Formation of an RNA Tertiary Structure

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg2+-RNA chelates could be responsible for a significant fraction of the Mg2+-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg2+ chelation is uncoupled from RNA folding and from unfavorable interactions with Mg2+ ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg2+- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg2+ concentration, and is partially resolved from the accumulation of Mg2+ in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg2+-RNA interaction free energies over the range of accessed Mg2+ concentrations. At 0.1 mM Mg2+ and 60 mM K+, specific site binding contributes similar to-3 kcal/mol of the total Mg2+ interaction free energy of similar to-13 kcal/mol from all sources; at higher Mg2+ concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg2+-RNA interactions.