Non-nearest neighbor effects on the thermodynamics of unfolding of a model mRNA pseudoknot

The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-even and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot. In phage T4, the pseudoknot is contained within 28 contiguous nucleotides which adopt a pseudocontinuous helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequivalent single-stranded loops of five and one nucleotide(s). These two loops cross the minor and major grooves of stems 1 and 2, respectively. In this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T4 gene 32 mRNA has been determined through analysis of dual-wave-length, equilibrium thermal melting profiles via application of a van't Hoff model based on multiple sequential, two-state transitions. The melting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration. Compensatory base-pair substitutions incorporated into helical stems 1 and 2 were used to assign the first low enthalpy, moderate tm melting transition to the denaturation of the short three to four base-pair stem 1, followed by unfolding of the larger seven base-pair stem 2. We find that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) strikingly uncouple the melting of stems 1 and 2, with the U10 substitution and the GA10 loop expansion more destabilizing than the G10 and C10 substitutions. A significant increase in the extent of cleavage by RNase T1 following the conserved G26 (the 3' nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix junction region in this mutant may be responsible, at least in part, for this uncoupling. In addition to a modest destabilization of stem 2, the major effect of deletion or nucleotide substitution in the 3' single-stranded tail is a destabilization of stem 1, a non-nearest neighbor tertiary structural effect, which may well be transmitted through an altered loop 1-core helix interaction. In contrast, truncation of the 5' tail has no effect on the stability of the molecule.