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

Lin, Ya-Hui; Chang, Kung-Yao

A comparison of theophylline-dependent −1 PRF activity between constructs using only one ligand-dependent regulator (theoOFF2-SARSPK versus Switch-1) indicated that the use of an upstream regulator provided better dynamic range for theophylline-dependent regulation than that of Switch-1 in vitro. However, the upstream attenuator seems to have reduced regulatory activity in cells and may be related to its different attenuation activities toward distinct downstream stimulators as observed previously (24) . Nevertheless, combining these two opposite regulators of −1 PRF helps further enhance the dynamic range of theophyllineresponsiveness both in vitro and in cells. Importantly, given that the only difference between the two aptamers used in theoOFF2 and Switch-1 is the base-pairing composition of their lower stems, this suggests that the same ligand can synergistically control a set of negative and positive −1 PRF regulators harboring homologous aptamers. Finally, the successful usage of polypeptides encoded by these regula-tory −1 PRF modules to create fused functional Venus proteins suggests that the modules can be inserted as a linker to bridge the coding sequences of two independent domains of a protein (such as the substrate-binding and catalytic domains of a specific enzyme) for regulation application.