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

Our previous NMR analysis of exchangeable imino protons of the SARS-CoV pseudoknot ( Figure 1A , wild-type pk) provided unequivocal evidence for the existence of Stem 3 (1). In the present study, secondary structure analysis by NMR provided further insight into the complex global architecture of the wild-type pk. To establish experimentally whether Stem 3 interacts with the two-stemmed H-type structure, we prepared a transcript lacking the base-paired region of Stem 3 while retaining the hexanucleotide 5 0 -ACU AGU-3 0 palindromic sequence ( Figure 1B , ÁS3 pk) and compared this construct with wild-type pk. The imino NOESY spectrum of the ÁS3 pk transcript is virtually identical to the wild-type pk native construct with a few marked exceptions. The missing sequential imino assignment path for the mutant ÁS3 pk is indicated by dashed lines in the superposition of wild-type pk and ÁS3 pk NOESY spectra shown in Figure 2A . For clarity, these nuclear Overhauser effect (NOE) connectivities including the characteristic G38-U59 wobble pair are highlighted in the schematic secondary structure of the three-stemmed pseudoknot structure (dashed box) and correspond to Stem 3. No significant chemical shift or linewidth changes can be observed for imino protons located outside of Stem 3. The only notable exception is the severely broadened crosspeak for the G14-C25 base-pair in the ÁS3 pk construct located in the vicinity of the Stem 1-Stem2 junction (Figure 2A , red box). A possible explanation for this observation is that a longer unpaired Loop 3 consisting of 12 (ÁS3 pk) rather than 3 nt (wild-type pk) may affect the degree of overrotation at the S1-S2 junction. Addressing this question in more detail would require a complete structure determination. At this stage, the large intrinsic linewidth of a 67-nt wild-type pk RNA in combination with severe line-broadening observed for NMR samples concentrated to >200 mM made this procedure unrealistic (data not shown). However, the overall comparison of exchangeable imino proton spectra of the wild-type pk with ÁS3 pk suggests that Stem 3 does not noticeably engage in stable tertiary interactions involving the two-stemmed H-type structure and likely constitutes an autonomous substructure within the frameshift signal.