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

To obtain direct structural information about the observed homodimers, a series of RNA transcripts were further investigated by NMR. Watson-Crick base pairing for the isolated Stem 3 RNA was confirmed by twodimensional nuclear NOESY spectra and a sequential imino walk is indicated in Figure 2B . As compared with the native S3L2 construct, a number of exchangeable imino resonances disappeared at 283 K in the NOESY spectrum of the asymmetric S3L2-ACUucc Loop 2 mutant. The missing sequential imino assignment path for the mutant S3L2-ACUucc corresponds to the upper portion of Stem 3 and is indicated by solid red lines in the superposition of S3L2 and S3L2-ACUucc imino NOESY investigation, we attempted to monitor the conversion by NMR with nucleotide-specific resolution in the presence of only potassium chloride. Therefore, we recorded a series of consecutive one-dimensional jump-return echo NMR spectra (Figure 7) to monitor S3L2 imino proton resonances in real-time. The overlapping G43 and U48 as well as G44 and G58 imino proton resonance assignments were confirmed using 2D heteronuclear 1 H, 15 N-HMQC correlations (Supplementary Figure S1 ). Dimer formation at 25 C was induced by addition of 125 mM potassium chloride to an NMR sample containing 250 mM S3L2 RNA and (interrupted) 1D spectra recorded over a period of >20 days. Figure 7 shows that half of the kissing complex is formed after $9 h and conversion progresses for >20 days before reaching a plateau characterized by a S3L2:(S3L2) 2 ratio of 2:8.