Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

To clarify the existences of theophylline-induced conformational switch and stem 2 formation, we tracked the distribution of single-stranded and duplex regions in free and theophylline-bound Switch-0 or Switch-1 RNA by limited ribonuclease T2 and V1 digestions, respectively (Figure 3 ). RibonucleaseV1 cleavages corresponding to the 3 -side of stem 1 for both free and theophylline-bound RNAs were in agreement with formation of stem 1 under both conditions for Switch-0 and Switch-1. Furthermore, V1 cleavage signals also occurred in the 5 -side of the upper stems of the aptamer domains but were reduced in the presence of theophylline in both Switch-0 and Switch-1, suggesting ligand-dependent rearrangement in the regions proximal to the binding-pockets. Consistent with these, reduced ribonu-cleaseT2 cleavage signals were also observed in bindingrelated nucleotides downstream of the upper stem upon theophylline treatment for both RNAs. However, major differences in T2 cleavage patterns between Switch-0 and Switch-1 appeared in the sequences covering 5 -sides of both binding pocket and lower stem of the aptamer domains, although the sequences forming binding-pockets are identical between the two RNAs. Furthermore, prominent T2 cleavages in these sequences observed for Switch-1 were reduced upon theophylline treatment and are consistent with theophylline-induced aptamer stabilization for Switch-1. Importantly, T2 cleavages also appeared in the stemloop junction of stem 1 in Switch-1 in the absence of theophylline, and were greatly reduced upon theophylline treatment. No similar T2 cleavage occurred in the corresponding Left OH − , A and T1 lanes represent denatured condition sequence markers corresponding to alkaline hydrolysis ladder, C/U residues-specific cut by RNase A and G residues-specific cut by RNase T1, respectively. Right T2, V1 and T1 lanes represent limited ribonuclease digestion by corresponding ribonuclease alone (−) or in the presence of either 1 mM theophylline (Theo) or 1 mM caffeine (Caf). Sequences corresponding to the 5 -side of stem 2, 5 -sides of lower aptamer stem/binding pocket and 3 -sides of binding pocket/lower aptamer stem are typed in the left from down to top (from 5 to 3 direction). Sequences identical between Switch-0 and Switch-1 are typed in capital, while the eight nucleotides different between them are typed in lower case. The predicted secondary structural elements along primary sequences are labeled in the right. Signals with reduced T2 and V1 cleavages in the presence of 1 mM theophylline are annotated by open and filled circle, respectively. region of Switch-0. Because these sequences constitute the 5 -side of stem 2 and are identical in both RNAs, these results are consistent with theophylline-induced formation of stem 2 in Switch-1. Together, these probing data are consistent with the existence of a theophylline-triggered conformation switch that leads to the formation of a pseudoknot.