Exploring the modular nature of riboswitches and RNA thermometers

Abstract Natural regulatory RNAs like riboswitches and RNA thermometers (RNAT) have considerable potential in synthetic biology. They are located in the 5′ untranslated region (UTR) of bacterial mRNAs and sense small molecules or changes in temperature, respectively. While riboswitches act on the level of transcription, translation or mRNA stability, all currently known RNATs regulate translation initiation. In this study, we explored the modularity of riboswitches and RNATs and obtained regulatory devices with novel functionalities. In a first approach, we established three riboswitch-RNAT systems conferring dual regulation of transcription and translation depending on the two triggers ligand binding and temperature sensing. These consecutive fusions control gene expression in vivo and can even orchestrate complex cellular behavior. In another approach, we designed two temperature-controlled riboswitches by the integration of an RNAT into a riboswitch aptamer domain. These ‘thermoswitches’ respond to the cognate ligand at low temperatures and are turned into a continuous on-state by a temperature upshift. They represent the first RNATs taking control of transcription. Overall, this study demonstrates that riboswitches and RNATs are ideal for engineering synthetic RNA regulators due to their modular behavior.

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