Tertiary Structure of an RNA Pseudoknot Is Stabilized by “Diffuse” Mg²⁺ Ions

Abstract

The aim of this study is to obtain a comprehensive experimental and theoretical description of the contributions of Mg2+ ions to the free energy of folding a pseudoknot RNA tertiary structure. A fluorescence method for measuring the effective concentration of Mg2+ in the presence of an RNA was used to study Mg2+-RNA interactions with both folded and partially unfolded forms of an RNA pseudoknot. These data established the excess number of Mg2+ ions accumulated by the folded or partially unfolded RNAs as a function of bulk Mg2+ concentration, from which free energies of Mg2+-RNA interactions were derived. Complementary thermal melting experiments were also used to obtain RNA-folding free energies. These experimental data were compared with the results of calculations based on the nonlinear Poisson-Boltzmann equation, which describes the interaction of "diffuse" (fully hydrated) Mg2+ ions with the different RNA forms. Good agreement between the calculations and experimental data suggests that essentially all of the Mg2+-induced stabilization of the native pseudoknot structure arises from the stronger interaction of diffuse ions with the folded tertiary structure compared to that with a partially unfolded state. It is unlikely that the stability of the RNA depends on dehydrated ions bound to specific sets of RNA ligands in the folded state. The data also suggest that the Mg2+-dependent free energy of folding is sensitive to factors that influence the ensemble of RNA conformations present in the partially unfolded state.

Continuum molecular electrostatics, salt effects, and counterion binding—A review of the Poisson–Boltzmann theory and its modifications

“…The NPB model of diffuse binding was also shown to reproduce correctly the experimental values for the change of the free energy of magnesium binding upon RNA folding. 93 In more advanced models, 50,94 the free energy of Mg binding to RNA is calculated in two steps which describe two modes of binding,…”

“…Because of the linearity of the Poisson equation in pure water, the total electrostatic potential of the fully charged RMg complex in pure water is / ¼ / can finally obtain an equation with expressions (93) and (94) on the left and right hand side, respectively. From the earlier equation one can conclude that the two approaches for calculating the electrostatic free energy of magnesium site binding represented with Eqs.…”

“…From the earlier equation one can conclude that the two approaches for calculating the electrostatic free energy of magnesium site binding represented with Eqs. (93) and (94) are equivalent. However, the two representations may not lead to the same results due to possible numerical errors.…”

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

“…However, the opposite conclusion has been drawn from fitting RNA folding transitions to PB predictions [70][71][72][73][74]. Ion-counting and SAXS experiments have provided a simple test for these effects and have revealed effects of ion size and valence that are not accounted for by PB theory, with deviations up to an order of magnitude in the systems investigated [58,75].…”

3 Importance of Diffuse Metal Ion Binding to RNA

“…The first key issue in the ion electrostatics is how diffuse ions are distributed around the RNA molecule [9][10][11][12][13][14][15][16][17][18][19]. There have been several useful experimental methods developed for the quantification of the ion atmosphere around nucleic acids.…”