Abstract: Riboswitches are important RNA structures in bacteria and some eukaryotes that can bind a ligand to toggle between conformations that allow or terminate transcription, translation, or splicing. We study folding kinetics and thermodynamics in the B. subtilis and T. maritima lysine riboswitches via single molecule TIRF microscopy. We show that riboswitch folding is exothermic with a large entropic cost, which we attribute to increased binding pocket rigidity upon lysine binding. Importantly, a minority of total bonds form between lysine and the RNA before folding is triggered, which indicates that lysine is recognized by the riboswitch via only a few crucial bonds. We examine the ionic cooperativity between potassium and lysine in the riboswitch to understand how potassium supports the folding process. Potassium dramatically decreases the unfolding rate and increases the folding rate for the riboswitch, an effect that is not replicated by Cs+ or Na+ ions as they may not be the correct size to mediate bonding between lysine and the RNA. This suggests that the riboswitch’s specificity for lysine could be tuned by the cations present and that riboswitch behavior in general may be influenced by more than just their target ligands. Last, we compare the T. maritima and B. subtilis riboswitches to understand the adaptations that compensate for their hyperthermophilic (80 °C) or mesophilic (40 °C) conditions. Both riboswitches must bind lysine before folding, have the same enthalpic changes upon folding, and undergo the same entropic changes, which imply extremely similar bond formation and conformational rearrangement upon folding. We determine that the higher G-C base pair content in the T. maritima riboswitch vs the B. subtilis riboswitch protects necessary pre-formed structures from melting at elevated temperatures, which demonstrates that adaptations to extremophilic conditions can rely on subtle differences in primary structure instead of changing ligand recognition and conformational rearrangement strategies.