Dataset Information

Allosteric mechanism of the V. vulnificus adenine riboswitch resolved by four-dimensional chemical mapping.

ABSTRACT: 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 (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, 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 LM2R 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.

SUBMITTER: Tian S

PROVIDER: S-EPMC5847336 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Publications

Allosteric mechanism of the <i>V. vulnificus</i> adenine riboswitch resolved by four-dimensional chemical mapping.

Tian Siqi S Kladwang Wipapat W Das Rhiju R

eLife 20180215

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<sup>2</sup>R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the ad ...[more]

PMID: 29446752

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Project description:Expression of a large fraction of genes in bacteria is controlled by riboswitches, which are found in the untranslated region of mRNA. Structurally riboswitches have a conserved aptamer domain to which a metabolite binds, resulting in a conformational change in the downstream expression platform. Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics (MD) and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in the add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for the pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

Dataset Information

Allosteric mechanism of the V. vulnificus adenine riboswitch resolved by four-dimensional chemical mapping.

Publications

Allosteric mechanism of the <i>V. vulnificus</i> adenine riboswitch resolved by four-dimensional chemical mapping.

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