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The three-dimensional conformations of non-coding RNAs underpin theirbiochemical functions but have largely eluded experimental characterization.Here, we report that integrating a classic mutation/rescue strategy withhigh-throughput chemical mapping enables rapid RNA structure inference withunusually strong validation. We revisit a paradigmatic 16S rRNA domain forwhich SHAPE (selective 2`-hydroxyl acylation with primer extension) suggested aconformational change between apo- and holo-ribosome conformations.Computational support estimates, data from alternative chemical probes, andmutate-and-map (M2) experiments expose limitations of prior methodology andinstead give a near-crystallographic secondary structure. Systematicinterrogation of single base pairs via a high-throughput mutation/rescueapproach then permits incisive validation and refinement of the M2-basedsecondary structure and further uncovers the functional conformation as anexcited state (25+/-5% population) accessible via a single-nucleotide registershift. These results correct an erroneous SHAPE inference of a ribosomalconformational change and suggest a general mutate-map-rescue approach fordissecting RNA dynamic structure landscapes.

Correcting a SHAPE-directed RNA structure by a mutate-map-rescue approach