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