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The structural interconversions that mediate the gene regulatory functions of RNA molecules may be different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM 2 R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via compensatory rescue evaluated by chemical mapping. These LM 2 R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and suggest additional structural heterogeneities that may be general across ligand-free riboswitches., Competing Interests: ST, WK, RD No competing interests declared, (© 2018, Tian et al.)