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 (-T Delta S-dock(double dagger)) and the overall entropic penalty (-T Delta S degrees(dock)) for docking, with essentially negligible effects on both the activation enthalpy (Delta H-dock(double dagger)) and overall exothermicity (Delta H degrees(dock)). 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 Delta H-dock(double dagger) and Delta H degrees(dock) 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.