Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the v iral life cycle. The 1.6 Angstrom crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson- Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na+. Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem te rtiary structural interactions which stabilize the molecule, contributing a net of Delta H approximate to - 30 kcal mol(-1) and Delta G(37)degrees, of -3.3 kcal mol(-1) to a total Delta H and aG(37)degrees, of -121 and -16 kc al mol(-1), respectively, at pH 6.0, 0.5 M K+ by DSC. Characterization of m utant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-trip le (pK(a) = 6.8), protonation of which contributes nearly -3.5 kcal mol(-1. ) in net stability in the presence of a wild-type loop 2. Substitution of t he nucleotides in loop 2 with uridine bases, which would eliminate the mino r groove triplex, destroys pseudoknot formation. An examination of the depe ndence of the monovalent ion and type on melting profiles suggests that ter tiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K+, NH4+ and Na+ on other s imple duplex and pseudoknotted RNAs. (C) 2000 Academic Press.