Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop-Receptor Tertiary Interaction

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 degrees C), from which a van't Hoff analysis yields the enthalpy (Delta H degrees) and entropy (Delta S degrees) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl2 and 100 mM NaCl, with excellent agreement between immobilized (Delta H degrees = -17.4 +/- 1.6 kcal/mol, and Delta S degrees = -56.2 +/- 5.4 cal mol(-1) K-1) and freely diffusing (Delta H degrees = -17.2 +/- 1.6 kcal/mol, and Delta S degrees = -55.9 +/- 5.2 cal mol(-1) K-1) species. Kinetic heterogeneity in the tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop-receptor interaction can account for similar to 60% of the Delta H degrees and Delta S degrees of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions.