title: Ligand binding to 2΄-deoxyguanosine sensing riboswitch in metabolic context.
abstract: The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit β of ribonucleotide reductase in Mesoplasma florum upon 2΄-deoxyguanosine binding. We characterized binding of 2΄-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and 'in-cell-like' conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. 'In-cell-like' environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under 'in-cell-like'-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2΄-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2΄-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2΄-deoxyguanosine, guanosine plays a role in riboswitch function in vivo. Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2΄-deoxyguanosine was not able to regulate gene expression.
authors.affiliation: Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 7, D-60438 Frankfurt/Main, Germany.
authors.forename: Yong-Boum
authors.initials: YB
authors.lastname: Kim
authors.affiliation: Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 7, D-60438 Frankfurt/Main, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Wacker
authors.affiliation: Department of Biology, Technical University Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.
authors.forename: Karl von
authors.initials: KV
authors.lastname: Laer
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Rogov
authors.affiliation: Department of Biology, Technical University Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Suess
authors.affiliation: Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 7, D-60438 Frankfurt/Main, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Schwalbe
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism; D001483:Base Sequence; D003849:Deoxyguanosine / Q000378:metabolism*; D017930:Genes, Reporter; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D055442:Metabolome; D009587:Nitrogen Isotopes; D009690:Nucleic Acid Conformation; D058928:Riboswitch; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D009587:Nitrogen Isotopes; D058928:Riboswitch; D001616:beta-Galactosidase; D003849:Deoxyguanosine