Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches

Abstract

AbstractThe ability to sense and respond to intracellular metabolite levels enables cells to adapt to environmental conditions. Many prokaryotes use riboswitches – structured RNA elements usually located in the 5’ untranslated region of mRNAs – to sense intracellular metabolites and respond by modulating gene expression. The corrinoid riboswitch class, which responds to adenosylcobalamin (coenzyme B12) and related metabolites, is among the most widespread in bacteria. The structural elements for corrinoid binding and the requirement for a kissing loop interaction between the aptamer and expression platform domains have been established for several corrinoid riboswitches. However, the conformational changes in the expression platform that modulate gene expression in response to corrinoid binding remain unknown. Here, we employ anin vivoGFP reporter system inBacillus subtilisto define alternative secondary structures in the expression platform of a corrinoid riboswitch fromPriestia megateriumby disrupting and restoring base-pairing interactions. Moreover, we report the discovery and characterization of the first riboswitch known to activate gene expression in response to corrinoids. In both cases, mutually exclusive RNA secondary structures are responsible for promoting or preventing the formation of an intrinsic transcription terminator in response to the corrinoid binding state of the aptamer domain. Knowledge of these regulatory mechanisms allowed us to develop synthetic corrinoid riboswitches that convert repressing riboswitches to riboswitches that robustly induce gene expression in response to corrinoids. Due to their high expression levels, low background, and over 100-fold level of induction, these synthetic riboswitches have potential use as biosensors or genetic tools.