Entropic origin of Mg2+-facilitated RNA folding

Mg2+ is essential for the proper folding and function of RNA, though the effect of Mg2+ concentration on the free energy, enthalpy, and entropy landscapes of RNA folding is unknown. This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop−receptor tertiary interaction as a function of [Mg2+]. These measurements yield the barrier and standard state enthalpies, entropies, and free energies for an RNA tertiary transition, in particular, revealing the thermodynamic origin of [Mg2+]-facilitated folding. Surprisingly, these studies reveal that increasing [Mg2+] promotes tetraloop–receptor interaction by reducing the entropic barrier () and the overall entropic penalty () for docking, with essentially negligible effects on both the activation enthalpy () and overall exothermicity (). These observations contrast with the conventional notion that increasing [Mg2+] facilitates folding by minimizing electrostatic repulsion of opposing RNA helices, which would incorrectly predict a decrease in and with [Mg2+]. Instead we propose that higher [Mg2+] can aid RNA folding by decreasing the entropic penalty of counterion uptake and by reducing disorder of the unfolded conformational ensemble.