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

The NMR line-broadening observed motivated a closer examination of the SARS pseudoknot and revealed a palindromic sequence in the loop capping Stem 3, designated Loop 2 (L2) ( Figure 1A ). To determine whether this palindromic sequence could mediate dimerization, Stem 3 transcripts ( Figure 1C , S3L2) were incubated at 37 C for 30 min in the presence of KCl and subjected to native PAGE. Lane 1 of Figure 3A shows two bands and demonstrates that S3L2 transcripts form homodimers when analysed at 4 C. To evaluate the role of the palindromic sequence for dimer formation, a series of mutations were generated ( Figure 1D -F) with the intention of altering the palindromic sequence ( Figure 1D ), replacing the entire Loop 2 ( Figure 1E ) or significantly reducing the size of both Stem 3 and Loop 2 ( Figure 1F ).