The helical junction region of a À1 frameshift stimulating hairpin-type mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) is characterized by a novel C27 Á (G7-C14) loop 2-stem 1 minor groove base triple, which is stacked on a C8+ Á (G12-C28) loop 1-stem 2 major groove base triple. Substitution of C27 with adenosine reduces frameshifting efficiency to a level just twofold above the slip-site alone. Here, we show that the global structure of the C27A ScYLV RNA is nearly indistinguishable from the wild-type counterpart, despite the fact that the helical junction region is altered and incorporates the anticipated isostructural A27 Á (G7-C14) minor groove base triple. This interaction mediates a 2.3-Å displacement of C8+ driven by an A27 N6-C8 + O2 hydrogen bond as part of an A (nÀ1) Á C + Á G-C n base quadruple. The helical junction regions of the C27A ScYLV and the beet western yellows virus (BWYV) pseudoknots are essentially superimposable, the latter of which contains an analogous A25 Á (G7-C14) minor groove base triple. These results reveal that the global ground-state structure is not strongly correlated with frameshift stimulation and point to a reduced thermodynamic stability and/or enhanced kinetic lability that derives from an altered helical junction architecture in the C27A ScYLV RNA as a significant determinant for setting frameshifting efficiencies in plant luteoviral mRNA pseudoknots.

The 39-untranslated region (UTR) of the group 2 coronavirus mouse hepatitis virus (MHV) genome contains a predicted bulged stem-loop (designated P0ab), a conserved cis-acting pseudoknot (PK), and a more distal stem-loop (designated P2). Base-pairing to create the pseudoknot-forming stem (P1 pk ) is mutually exclusive with formation of stem P0a at the base of the bulged stemloop; as a result, the two structures cannot be present simultaneously. Herein, we use thermodynamic methods to evaluate the ability of individual subdomains of the 39 UTR to adopt a pseudoknotted conformation. We find that an RNA capable of forming only the predicted PK (58 nt; 39 nucleotides 241-185) adopts the P2 stem-loop with little evidence for P1 pk pairing in 0. , 100 mM K + ), and unfolded at 37°C. Similar findings characterize an RNA 59 extended through the P0b helix only (89 nt; 294-185). In contrast, an RNA capable of forming either the P0a helix or the pseudoknot (97 nt; 301-185) forms no P1 pk helix. Thermal unfolding simulations are fully consistent with these experimental findings. These data reveal that the PK forms weakly and only when the competing doublehairpin structure cannot form; in the UTR RNA, the double hairpin is the predominant conformer under all solution conditions.