Mg 2+ –RNA interaction free energies and their relationship to the folding of RNA tertiary structures

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Abstract
Mg 2+ ions are very effective at stabilizing tertiary structures in RNAs. In most cases, folding of an RNA is so strongly coupled to its interactions with Mg 2+ that it is difficult to separate free energies of Mg 2+ – RNA interactions from the intrinsic free energy of RNA folding. To devise quantitative models accounting for this phenomenon of Mg 2+ -induced RNA folding, it is necessary to independently determine Mg 2+ –RNA interaction free energies for folded and unfolded RNA forms. In this work, the energetics of Mg 2+ –RNA interactions are derived from an assay that measures the effective concentration of Mg 2+ in the presence of RNA. These measurements are used with other measures of RNA stability to develop an overall picture of the energetics of Mg 2+ -induced RNA folding. Two different RNAs are discussed, a pseudoknot and an rRNA fragment. Both RNAs interact strongly with Mg 2+ when partially unfolded, but the two folded RNAs differ dramatically in their inherent stability in the absence of Mg 2+ and in the free energy of their interactions with Mg 2+ . From these results, it appears that any comprehensive framework for understanding Mg 2+ -induced stabilization of RNA will have to ( i ) take into account the interactions of ions with the partially unfolded RNAs and ( ii ) identify factors responsible for the widely different strengths with which folded tertiary structures interact with Mg 2+ .