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

Lin, Ya-Hui; Chang, Kung-Yao

With a Kd value of 1.31 M for interaction between theophylline and Switch-1 RNA, it took at least 100 M of theophylline to start observing −1 PRF stimulation in vitro ( Figure 4B -D) and 1 mM of theophylline to reach −1 PRF efficiency rivals that of viral stimulator in cellular assay ( Figure 4E ). This suggests that the artificial mammalian riboswitch needs to be fully bound by the ligand to effectively stimulate −1 PRF and is consistent with the theophylline concentration required to fully saturate Switch-1 RNA in affinity measurement plot (Supplement Figure S2C ). By contrast, the higher concentration required in cellular condition could be due to the cellular uptake efficiency of theophylline and reduced theophylline-binding affinity of Switch-1 at 37 • C. Indeed, studies in bacterial riboswitch regulation of transcription termination also indicated that much higher metabolite concentration is required to observe effective transcription termination in vitro (41) because a riboswitch is composed of an aptamer and an RNAbased gene expression platform. Thus, effective ligand concentration required for gene expression regulation in both bacterial and mammalian riboswitches is much higher than the Kd value that saturates 50% of its receptor, and could be varied to different extents due to the involvement of different expression platforms.