title: High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states.
abstract: The three-dimensional conformations of noncoding 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 16S rRNA domain for which SHAPE (selective 2'-hydroxyl acylation with primer extension) and limited mutational analysis suggested a conformational change between apo- and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M(2)) experiments highlight issues 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 M(2)-based secondary structure. The data further uncover the functional conformation as an excited state (20 ± 10% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change, expose critical limitations of conventional structure mapping methods, and illustrate practical steps for more incisively dissecting RNA dynamic structure landscapes.
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Siqi
authors.initials: S
authors.lastname: Tian
authors.affiliation: Biomedical Informatics Program, Stanford University, Stanford, California 94305, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Cordero
authors.affiliation: Department of Physics, Stanford University, Stanford, California 94305, USA.
authors.forename: Wipapat
authors.initials: W
authors.lastname: Kladwang
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA Biomedical Informatics Program, Stanford University, Stanford, California 94305, USA Department of Physics, Stanford University, Stanford, California 94305, USA rhiju@stanford.edu.
authors.forename: Rhiju
authors.initials: R
authors.lastname: Das
mesh_terms: D057166:High-Throughput Screening Assays; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D059370:RNA Folding; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D012336:RNA, Ribosomal, 16S
keywords: RNA folding; SHAPE; compensatory rescue; mutate-and-map; ribosome; secondary structure