Thermodynamics of RNA folding in a conserved ribosomal RNA domain

A small, 58 nt domain of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1108) is a highly conserved junction of three helices whose secondary structure has been established by phylogenetic comparisons. To detect any contributions of additional tertiary interactions, the thermal denaturation of the rRNA domain was followed by either UV hyperchromicity or calorimetry in buffers containing a wide range of Mg2+ concentrations. Several smaller fragments corresponding to two different hairpin stem-loop structures within the domain were also synthesized and melted for comparison with the larger molecule. A model of the secondary structure unfolding was devised, based on measured enthalpies and melting temperatures of the component hairpins and tabulated parameters of base-pair stacking and loop closure. The model closely simulates the observed melting data when three additional factors are included: two parameters to account for coaxial stackings within a junction of helices, and a set of undefined "tertiary" interactions that unfolds before the secondary structure and is preferentially stabilized by Mg2+. A critical feature of this model is a conserved pair, U1082/A1086, that is within the junction loop and hypothesized to stack with an adjacent helix. The model correctly predicts the effects of disrupting this pair in a U1086 sequence variant. Although the set of "tertiary" interactions contributes a significant fraction of the RNA unfolding enthalpy (delta H approximately 25 kcal/mol, out of 180 kcal/mol total), its overall stability is marginal at 37 degrees C.