期刊
RNA
卷 17, 期 9, 页码 1747-1759出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.2816711
关键词
coronavirus replication; RNA pseudoknot; RNA folding; conformational switch; SARS-CoV; mouse hepatitis virus
资金
- NIH [AI040187, AI067416, GM063732]
- NSF [MCB0920067, MCB0920411]
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 39 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 Mg2+; as Mg2+ 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, Delta G(25) approximate to 1.2 +/- 0.3 kcal/mol (5.0 mM Mg2+, 100 mM K+), and unfolded at 37 degrees 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.
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