The thermodynamic origin of the stability of a thermophilic ribozyme

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investi gated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scatteri ng, and circular dichroism. Both RNAs require Mg2+ to fold to their native structures that are very similar. The stability is measured as a function o f Mg2+ and urea concentrations at different temperatures. The enhanced stab ility of the thermophilic ribozyme primarily is derived from a tremendous i ncrease in the amount of structure formed in the ultimate folding transitio n. This increase in structure formation and cooperativity arises because th e penultimate and the ultimate folding transitions in the mesophilic ribozy me become linked into a single transition in the folding of the thermophili c ribozyme. Therefore, the starting point, or reference state, for the tran sition to the native, functional thermophilic ribozyme is significantly les s structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected na tive interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to ach ieve higher functional stability for tertiary RNAs is fundamentally differe nt from that commonly proposed to explain the increased stability of thermo philic proteins.