title: Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure.
abstract: 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.
authors.affiliation: Department of Biophysics, Johns Hopkins University Baltimore, Maryland 21218, United States.
authors.forename: Desirae
authors.initials: D
authors.lastname: Leipply
authors.forename: David E
authors.initials: DE
authors.lastname: Draper
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D059370:RNA Folding; D012335:RNA, Ribosomal / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D012335:RNA, Ribosomal; D012313:RNA; D008274:Magnesium