RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Ishimaru, Daniella; Plant, Ewan P.; Sims, Amy C.; Yount, Boyd L.; Roth, Braden M.; Eldho, Nadukkudy V.; Pérez-Alvarado, Gabriela C.; Armbruster, David W.; Baric, Ralph S.; Dinman, Jonathan D.; Taylor, Deborah R.; Hennig, Mirko

No evidence for further progression to a potential extended duplex structure could be detected. Curiously, the replacement of the 9-nt loop L2 with the stable 5 0 -cuug-3 0 tetraloop (S3-cuug, Figure 1E ) designed to stabilize hairpin formation generated a transcript that efficiently dimerized ( Figure 3A, lane 2) . The imino assignments through the stem could be easily followed based on the resonance assignments and NOE patterns observed in S3L2 (Supplementary Figure S2) . Close inspection of the sequential assignments and NOE patterns revealed the existence of tandem U-U wobble pairs involving the two uridine nucleotides of the tetraloop. In addition, substantial line broadening was observed consistent with extended duplex formation of S3-cuug transcripts (Supplementary Figure S2 ). Subsequently, we examined the NOESY spectra of the larger, three-stemmed 5 0 -cuug-3 0 -tetraloop containing SARS mutants S3-2bp-cuug ( Figure 1F ) and S3-cuug pk that were subjected to functional frameshifting analysis. We confirmed the formation of the tandem U-U wobble pairs by 2D NOESY. The NOE patterns for the U-U wobbles are an almost perfect subspectrum of the same region of the NOESY for the isolated S3-cuug transcripts (Supplementary Figure S3) . Other exchangeable protons in the Stem 1 and Stem 2 regions comprising the H-type pseudoknot overlay with the corresponding imino protons observed in the context of wild-type pk. The excellent agreement in the overlay of the imino connectivities traced by the NOESY experiments indicates that the stable 5 0 -cuug-3 0 tetraloop capping an otherwise wild-type Stem 3 facilitates the formation of extended duplex structures in the context of larger S3-2bp-cuug and S3-cuug pk mutants and locally retains the same structure as S3-cuug in isolation.