Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Authors

  • Dr. Julia E. Weigand,

    1. Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany)
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    • These authors contributed equally to this work.

  • Sina R. Gottstein-Schmidtke,

    1. Institute of Molecular Biosciences, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany)
    2. Center of Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany)
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    • These authors contributed equally to this work.

  • Shemsi Demolli,

    1. Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany)
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  • Florian Groher,

    1. Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany)
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  • Dr. Elke Duchardt-Ferner,

    1. Institute of Molecular Biosciences, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany)
    2. Center of Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany)
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  • Prof. Dr. Jens Wöhnert,

    1. Institute of Molecular Biosciences, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany)
    2. Center of Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany)
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  • Prof. Dr. Beatrix Suess

    Corresponding author
    1. Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany)
    • Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany)===

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Abstract

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA–ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence–activity relationships that could not initially be explained on the basis of the structure of the aptamer–ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.

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