Novel Heat-Promoted Folding Dynamics of the yybP-ykoY Manganese Riboswitch: Kinetic and Thermodynamic Studies at the Single-Molecule Level.

Riboswitches are highly structured RNA elements that regulate gene expressions by undergoing conformational changes in response to their cognate ligands. Such regulatory strategies are particularly prevalent among bacteria, which need to be evolutionarily responsive to thermal fluctuations in the surrounding environment, for example, generating extremophiles evolved to survive anomalously high or low temperatures. As a consequence, the response of such riboswitches to thermal stress becomes of considerable interest. In this study, the temperature-dependent folding kinetics and thermodynamics of the manganese riboswitch ( yybP-ykoY) is studied by single-molecule FRET spectroscopy under external thermal control. Surprisingly, the folding of the manganese riboswitch is found to be strongly promoted by temperature. Detailed thermodynamic analysis of the equilibrium and forward/reverse kinetic rate constants reveal folding to be a strongly endothermic process (Δ H) made feasible by an overall entropic lowering (- TΔ S) in free energy. This is in contrast to a more typical picture of RNA folding achieving a more compact, highly ordered state (Δ S) and clearly speaks to the significant role of solvent/cation reorganization in the folding thermodynamics. With the help of the transition-state theory, free energy landscapes for the manganese riboswitch are constructed from the temperature-dependent kinetic data, revealing two distinctive folding mechanisms promoted by Mg and Mn, respectively. It is speculated that this unconventional temperature dependence for folding of the manganese riboswitch may reflect evolution of bacterial gene regulation strategies to survive environments with large-temperature variations.