Folding of an mRNA Pseudoknot Required for Stop Codon Readthrough: Effects of Mono- and Divalent Ions on Stability

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Abstract
Unfolding of an mRNA pseudoknot that induces ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus has been studied by UV hyperchromicity and calorimetry. The pseudoknot melts in two steps, corresponding to its two helical stems. The total enthalpy of denaturation is170 kcal/mol, approximately the value expected for the secondary structure. At low salt concentrations ( Na+ > K+ > Rb+ > Cs+) and by NH4+; the same preferences are exhibited by one of the stems in the context of a hairpin. Divalent metal ions are not required to fold the pseudoknot but do stabilize it further. To examine divalent ion effects over a wide concentration range, urea was used to lower the RNA unfolding temperature and was shown not to affect characteristics of the pseudoknot unfolding in other respects. The pseudoknot binds divalent ions somewhat more tightly than a hairpin but shows only weak selectivity for different size ions. It is suggested that a region of "intermediate" divalent ion binding affinity, in between highly ligated specific sites and purely delocalized ion binding in character, is created by the pseudoknot fold but that nonspecific, delocalized ion binding contributes at least half the free energy of pseudoknot stabilization by Mg2+. Functional RNAs are generally highly folded, compact structures whose stabilities are sensitive to the concentrations and types of ions present. Two basic types of ion interactions with RNAs may be imagined. The high negative charge of