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 examine the influence of temperature on dimer formation, S3L2 RNAs were incubated in the presence of MgCl 2 at 25 C and 37 C (Figures 4C and 4D ). Aliquots at various time-points were collected and stored at À20 C until all samples were collectively analysed by native PAGE. As shown in Figure 4D , S3L2 dimers readily formed at physiological temperatures (T ½ = 48 ± 12 min), while dimer formation was considerably slower at room temperature (T ½ = 19.8 ± 6.5 h; Figure 4C ). We also quantified the concentration dependence of S3L2 dimerization, in the presence of MgCl 2 , and determined the dissociation constant for the dimer to be 2.6 ± 0.15 mM at 37 C ( Figure 4F ). Taken together these results suggest that Stem 3/Loop 2 readily dimerizes under physiological conditions, i.e. at 37 C in the presence of Figure 2 . NMR secondary structure comparison of wild-type SARS-CoV pk, ÁS3 pk, S3 and S3L2-ACUucc mutants. (a) Imino regions of 2D 1 H, 1 H-NOESY experiments collected on wild-type SARS-CoV pk (black contours) and ÁS3 pk mutant (red contours), respectively. Dashed black lines show the imino proton walk in the S3 stem. The base-paired region of S3 is deleted in the ÁS3 pk mutant; however, L2 is left intact. Solid red lines show the sequential NOE correlations involving the imino proton U26 located in S1, and the red box highlights the cross peak connecting imino protons U26 and G14 adjacent to the S1-S2 junction, which is absent in the ÁS3 pk mutant. Only the last two digits of the wild-type sequence numbering are used for clarity. The schematic SARS-CoV pk inset highlights the corresponding S3 stem (dashed box) as well as the G14-C25 basepair location in S1 (solid red box). (b) Imino regions of 2D 1 H, 1 H-NOESY experiments collected on wild-type SARS S3 (black contours) and the S3L2-ACUucc mutant (red contours), respectively. Dashed black lines show the imino proton walk in the lower portion of the S3 stem. Solid red lines highlight the sequential cross peaks in the upper portion of S3 correlating imino protons G55, U54 and G43 adjacent to L2, which are broadened beyond detection in the S3L2-ACUucc mutant. The schematic SARS S3L2 inset highlights the corresponding lower S3 stem (dashed box) as well as the base-paired region in the upper S3 stem (solid red box). 5 mM MgCl 2 , and that homodimers tolerate a broad range of ionic strengths.