Thermodynamics of folding a pseudoknotted mRNA fragment

A sequence in the leader and first gene of the Escherichia coli alpha mRNA folds into a complex pseudoknot structure that is required for binding of a translational repressor. The thermal denaturation of a 112 nt RNA containing this structure has been followed by calorimetry and UV hyperchromicity. To determine the partially folded intermediates in unfolding, the denaturation of 13 mutants and of several fragments with successive deletions of helices were investigated as well. An unfolding pathway with seven states is proposed as the simplest mechanism that accounts for the data, and has several implications. (1) The lowest temperature transition appears only in the presence of moderate concentrations of Mg2+ or high concentrations of K+ (delta H approximately 45 kcal/mol), and is the unfolding of tertiary structures, rather than secondary structure. Under some conditions it is destabilized by increasing salt concentration. (2) Two of the intermediates unfolding at higher temperature must have non-canonical or tertiary interactions in addition to the known secondary structure. (3) Two alternative structures compete for formation of the complete pseudoknot, and form as the pseudoknot unfolds. Thus structures not present in the completely folded pseudoknot affect the overall thermodynamics, and probably the kinetics, of unfolding. (4) Approximately 16 kcal/mol of free energy is required to completely expose the coding region to ribosomes at 37 degrees C, though approximately 6.5 kcal/mol is regained by refolding of upstream regions after the pseudoknot is unfolded. The substantial energy needed to unfold the pseudoknot may affect the rate of translation from this ribosome binding site. A simple model of RNA folding in which an optimum secondary structure forms first, followed by tertiary interactions that further stabilize the secondary structure, does not hold in this RNA.