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

Abstract

Retroviruses employ −1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 Å 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 tertiary structural interactions which stabilize the molecule, contributing a net of ΔH≈−30 kcal mol−1 and ΔG°37 of −3.3 kcal mol−1 to a total ΔH and ΔG°37 of −121 and −16 kcal mol−1, respectively, at pH 6.0, 0.5 M K+ by DSC. Characterization of mutant RNAs supports the presence of a C8+·G12-C26 loop 1-stem 2 base-triple (pKa=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 the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K+, NH4+ and Na+ on other simple duplex and pseudoknotted RNAs.