Design and discovery of 'tug-of-war' riboswitches

Riboswitches are compact regulatory RNAs that modulate gene expression by switching between mutually exclusive structures in response to binding diverse signalling molecules. Riboswitches that regulate transcription have been shown to function through sequence overlap between their ligand binding aptamer domain and their regulatory expression platform, a constraint that limits their engineering for biotechnological applications. Here we describe a new, modular, transcriptional riboswitch architecture, termed "tug-of-war" (TOW), that removes this constraint, based on the discovery that ligand-stabilized RNA structures positioned immediately upstream of intrinsic terminators suppress termination. Using diverse aptamers, we create four distinct TOW riboswitches by pairing aptamer domains with terminators, and discover design principles for tuning their function. We demonstrate that upstream secondary and tertiary RNA structures generally inhibit intrinsic termination, consistent with a model of TOW antitermination dependant upon steric competition for occupancy of the RNA polymerase exit channel. Finally, we discover the TOW architecture to be present in nature through bioinformatic analysis and experimental validation, finding that ~10% of natural ZTP riboswitches - nearly all in gram-positive bacteria - employ the TOW architecture. Together, these findings define a new mechanism of nascent RNA-mediated antitermination that may be useful for searching for riboswitches in other domains of life, and establish TOW riboswitches as a highly modular platform for engineering transcriptional RNA regulators.