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

Lin, Ya-Hui; Chang, Kung-Yao

Next, we measured the in vitro −1 PRF activity of Switch-1 ( Figure 4A) in the presence of different amounts of theophylline using reticulocyte lysate. The −1 PRF activity of Switch-1 responded to theophylline treatment in a dosagedependent manner and was virtually non-responsive to 1 mM caffeine. The mutant construct with theophyllinebinding pocket disrupted (Switch-1M1) possessed minor (39) in 293T cell with or without 1 mM theophylline. −1 PRF activity was calculated by calibrating with the dual-luciferase activity of p2luci as a read-through control (23) . Value for each bar is the mean of three independent experiments with standard error of the mean. (F) Comparison of relative −1 PRF activity of Switch-1 and SAH-PK (8, 24) in 293T cell toward cognate ligand variation. A total of 1 M of Adox, an SAH hydrolyase inhibitor was used to increase concentration of SAH in 293T cells (24) . Relative −1 PRF activity was calculated by calibrating with the dual-luciferase activity of P2luci as a read-through control while the drug-free activity was treated as 1 (in gray). Value for each bar is the mean of three independent experiments with standard error of the mean. For all panels, P-values were determined by a Student's t-test with P-value < 0.05 designated by an '*'. increment of frameshifting activity in 1 mM theophylline ( Figure 4B and C) . In addition, dual-luciferase based −1 PRF activity obtained from 293T cells transfected by Switch-1 containing reporter possessed a similar dosagedependent trend toward theophylline as that of the in vitro analysis, whereas cells transfected by Switch-1M1 reporter lost theophylline-dependency for −1 PRF activity ( Figure 4D ). Finally, side-by-side comparison indicates that the −1 PRF efficiency of Switch-1 in 1 mM theophylline rivals those of MMTV and SRV −1 PRF stimulators (39) (Figure 4E) , while the dynamic range of theophylline-dependent frameshifting stimulation is close to the level of SAH-PK toward SAH variation (8,24) ( Figure 4F) . Collectively, the results of probing and functional assays demonstrate that Switch-1 is a bona-fide mammalian riboswitch using −1 PRF as the expression platform.