NON-NEAREST NEIGHBOR EFFECTS ON THE THERMODYNAMICS OF UNFOLDING OF A MODEL MESSENGER-RNA PSEUDOKNOT

The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-ev en and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot. In phage T4, the pseudoknot is c ontained within 28 contiguous nucleotides which adopt a pseudocontinuo us helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequi valent single-stranded loops of five and one nucleotide(s). These two loops cross the minor and major grooves of stems 1 and 2, respectively . Ln this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of t he T4 gene 32 mRNA has been determined through analysis of dual-wavele ngth, equilibrium thermal melting profiles via application of a van't Hoff model based on multiple sequential, two-state transitions. The me lting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration. Com pensatory base-pair substitutions incorporated into helical stems 1 an d 2 were used to assign the first low enthalpy, moderate t(m) melting transition to the denaturation of the short three to four base-pair st em 1, followed by unfolding of the larger seven base-pair stem 2. We f ind that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) st rikingly uncouple the melting of stems 1 and 2, with the U10 substitut ion and the GA10 loop expansion more destabilizing than the G10 and CI O substitutions. A significant increase in the extent of cleavage by R Nase T-1 following the conserved G26 (the 3' nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix ju nction region in this mutant may be responsible, at least in part, for this uncoupling. In addition to a modest destabilization of stem 2, t he major effect of deletion or nucleotide substitution in the 3' singl e-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. (C) 1998 Acade mic Press Limited.