Allosteric Mechanism of the V. vulnificus Adenine Riboswitch Resolved by Four-dimensional Chemical Mapping

Abstract

The structural logic that define the functions of gene regulatory RNA molecules may be radically different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied RNA model systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM2R), 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 including its gene expression platform, 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 high-throughput compensatory rescue experiments. These LM2R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and reveal new structural heterogeneities that may be general across ligand-free riboswitches.