Tuning strand displacement kinetics enables programmable ZTP riboswitch dynamic range in vivo

A large body of work has shown that transcriptional riboswitches function through internal strand displacement mechanisms that guide the formation of alternative structures which drive regulatory outcomes. Here, we sought to investigate this phenomenon using the Clostridium beijerinckii pfl ZTP riboswitch as a model system. Using functional mutagenesis with Escherichia coli gene expression assays, we show that mutations designed to slow strand displacement of the expression platform enable precise tuning of riboswitch dynamic range (2.4-34-fold), depending on the type of kinetic barrier introduced, and the position of the barrier relative to the strand displacement nucleation site. We also show that expression platforms from a range of different Clostridium ZTP riboswitches contain sequences that impose these barriers to affect dynamic range in these different contexts. Finally, we use sequence design to flip the regulatory logic of the riboswitch to create a transcriptional OFF-switch, and show that the same barriers to strand displacement tune dynamic range in this synthetic context. Together, our findings further elucidate how strand displacement can be manipulated to alter the riboswitch decision landscape, suggesting that this could be a mechanism by which evolution tunes riboswitch sequence, and providing an approach to optimize synthetic riboswitches for biotechnology applications.

Automated summary

Using the Clostridium beijerinckii pfl ZTP riboswitch as a model system, this study demonstrates that slowing down strand displacement can enable precise tuning of the riboswitch dynamic range. Various sequence barriers in the expression platforms of different Clostridium ZTP riboswitches were found to affect the dynamic range in different contexts. Additionally, the study shows that the same barriers can tune the dynamic range in a synthetic context, further elucidating how strand displacement can be manipulated to alter the riboswitch decision landscape.