A conserved RNA pseudoknot in a putative molecular switch domain of the 3'-untranslated region of coronaviruses is only marginally stable

The 3′-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 (P1pk) is mutually exclusive with formation of stem P0a at the base of the bulged stem–loop; as a result, the two structures cannot be present simultaneously. Herein, we use thermodynamic methods to evaluate the ability of individual subdomains of the 3′ UTR to adopt a pseudoknotted conformation. We find that an RNA capable of forming only the predicted PK (58 nt; 3′ nucleotides 241–185) adopts the P2 stem–loop with little evidence for P1pk pairing in 0.1 M KCl and the absence of Mg2+; as Mg2+ or 1 M KCl is added, a new thermal unfolding transition is induced and assignable to P1pk pairing. The P1pk helix is only marginally stable, ΔG25 ≈ 1.2 ± 0.3 kcal/mol (5.0 mM Mg2+, 100 mM K+), and unfolded at 37°C. Similar findings characterize an RNA 5′ 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 P1pk helix. Thermal unfolding simulations are fully consistent with these experimental findings. These data reveal that the PK forms weakly and only when the competing double-hairpin structure cannot form; in the UTR RNA, the double hairpin is the predominant conformer under all solution conditions.