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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 Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-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 Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) 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; Mg(2+)- 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 Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ∼-3 kcal/mol of the total Mg(2+) interaction free energy of ∼-13 kcal/mol from all sources; at higher Mg(2+) 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 Mg(2+)-RNA interactions.