Figure 1.

Equilibrium folding models for a thermophilic (C^thermo) and a mesophilic (C^meso) RNA. (A) The Mg²⁺-dependent equilibrium folding for both C^thermo and C^meso has two identical intermediates, I_t and I_m. Fractional population of each species is shown. (B) The free energy changes for each transition. The solid lines represent the experimentally determined ΔG as a function of Mg²⁺ concentration as defined by ΔG = −RT ln([N]/([I_t] + [I_m])). (C) Free energy diagrams at Mg²⁺ concentrations where both ribozymes have the same functional stability, ΔG_I-to-N = −2.0 kcal/mol, corresponding to 0.16 and 3.5 mM Mg²⁺ for the thermophilic and the mesophilic ribozyme, respectively. Near the folding transition, the I_t state is less stable than the I_m state, so that the latter largely defines the functional stability of the native mesophilic ribozyme. The relative stability of the intermediates is switched so that the I_t is the defining species for the stability of the thermophilic ribozyme. For C^meso, an increase in the stability of I_m would shift it and the native species down by the same amount, and not alter the functional stability.