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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 \u201ctug-of-war\u201d (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 \u223c10% of natural ZTP riboswitches \u2013 nearly all in gram-positive bacteria \u2013 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.<\/jats:p>","DOI":"10.64898\/2026.06.17.733018","type":"posted-content","created":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T10:30:15Z","timestamp":1781865015000},"source":"Crossref","is-referenced-by-count":0,"title":["Design and discovery of \u2018tug-of-war\u2019 riboswitches"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1987-0578","authenticated-orcid":false,"given":"David Z.","family":"Bushhouse","sequence":"first","affiliation":[{"name":"Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan 49503, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayu","family":"Fu","sequence":"additional","affiliation":[{"name":"Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0619-6505","authenticated-orcid":false,"given":"Julius B.","family":"Lucks","sequence":"additional","affiliation":[{"name":"Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Center for Water Research, Northwestern University, Evanston, Illinois 60208, USA"},{"name":"Center for Engineering Sustainability and Resilience, Northwestern University, Evanston, Illinois 60208, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026062202001401000_2026.06.17.733018v1.1","doi-asserted-by":"publisher","DOI":"10.1261\/rna.061234.117"},{"key":"2026062202001401000_2026.06.17.733018v1.2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.1c00765"},{"key":"2026062202001401000_2026.06.17.733018v1.3","doi-asserted-by":"crossref","first-page":"607158","DOI":"10.3389\/fmolb.2020.607158","article-title":"Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control","volume":"7","year":"2021","journal-title":"Front. 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