Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the jun ction of the two helical stems required for efficient frameshifting activit y, The thermodynamics of folding of the MMTV vpk pseudoknot have been compa red with a structurally homologous mutant RNA containing a G . U to G-C sub stitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13C Delta A14 RNA). Dual wavelength optical melting and d ifferential scanning calorimetry reveal that the unpaired adenosine contrib utes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (Delta G(37)degrees) at 1 M NaCl. This stability increment d erives from a favorable enthalpy contribution to the stability Delta Delta H = 6.6 (+/-2.1) kcal mol(-1) with Delta Delta G(37)degrees comparable to t hat predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G . U base pair. Group 1A monovalent ions, NH4+, Mg-2(+), and Co(NH3)(6 )(3+) ions stabilize the A14 and Delta A14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these mol ecules do not derive from a differential or specific accumulation of ions i n the A14 versus Delta A14 pseudoknots. Knowledge of this free energy contr ibution may facilitate the prediction of RNA pseudoknot formation from prim ary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).