Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed-1 ribosomal frameshifting

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseu doknot from the gag-pro junction of mouse intracisternal A-type particles ( mIAP), an endogenous retrovirus, has been determined through analysis of du al optical wavelength, equilibrium thermal melting profiles and differentia l scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot propose d to have structural features in common with the gag-pro frameshifting pseu doknots from simian retrovirus-l (SRV-1) and mouse mammary tumor virus (MMT V). Ln particular, the mIAP pseudoknot is proposed to contain an unpaired a denosine base at the junction of the two helical stems (A15), as well as on e in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins a nd compensatory base-pair substitutions incorporated into helical stem 2 wa s used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in t wo closely coupled low-enthalpy transitions at low t(m) in which the stem 3 ' to A35, unfolds first, followed by unfolding of the remainder of the heli cal stem. The third unfolding transition is associated with some type of st acking interactions in the stem 1 hairpin loop not present in the pseudokno t. The fourth transition is assigned to unfolding of stem 1. in all RNAs in vestigated, Delta H-vH approximate to Delta H-cal, suggesting that Delta C- p for unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the ste m 2 unfolding transition in the context of both the wild-type and Delta A35 mutant RNAs only slightly, by Delta Delta G degrees approximate to 1 kcal mol(-1) (at 37 degrees C). The Delta A15 RNA is considerably more susceptib le to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabil ization of the molecule. interestingly, substitution of the nine loop 2 nuc leotides with uridine residues induces a more pronounced destabilization of the molecule (Delta Delta G degrees approximate to 2.0 kcal mol(-1)), a lo ng-range, non-nearest neighbor effect. These findings provide the thermodyn amic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability. (C) 1998 Ac ademic Press.