title: A conserved RNA pseudoknot in a putative molecular switch domain of the 3'-untranslated region of coronaviruses is only marginally stable.
abstract: 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 (P1(pk)) 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 P1(pk) pairing in 0.1 M KCl and the absence of Mg(2+); as Mg(2+) or 1 M KCl is added, a new thermal unfolding transition is induced and assignable to P1(pk) pairing. The P1(pk) helix is only marginally stable, ΔG(25) ≈ 1.2 ± 0.3 kcal/mol (5.0 mM Mg(2+), 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 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 double-hairpin structure cannot form; in the UTR RNA, the double hairpin is the predominant conformer under all solution conditions.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Suzanne N
authors.initials: SN
authors.lastname: Stammler
authors.forename: Song
authors.initials: S
authors.lastname: Cao
authors.forename: Shi-Jie
authors.initials: SJ
authors.lastname: Chen
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
mesh_terms: D020413:3' Untranslated Regions; D020029:Base Pairing; D001483:Base Sequence; D016679:Genome, Viral; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000502:physiology; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics; D013816:Thermodynamics; D014779:Virus Replication / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D012367:RNA, Viral