title: Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.
abstract: RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.
authors.affiliation: Fachbereich Physik, Universität Osnabrück, 49069 Osnabrück, Germany.
authors.forename: Dorith
authors.initials: D
authors.lastname: Wunnicke
authors.forename: Denise
authors.initials: D
authors.lastname: Strohbach
authors.forename: Julia E
authors.initials: JE
authors.lastname: Weigand
authors.forename: Bettina
authors.initials: B
authors.lastname: Appel
authors.forename: Emiliano
authors.initials: E
authors.lastname: Feresin
authors.forename: Beatrix
authors.initials: B
authors.lastname: Suess
authors.forename: Sabine
authors.initials: S
authors.lastname: Müller
authors.forename: Heinz-Jürgen
authors.initials: HJ
authors.lastname: Steinhoff
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D058928:Riboswitch / Q000235:genetics*; D013113:Spin Labels; D013752:Tetracycline / Q000494:pharmacology*; D013816:Thermodynamics
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D011500:Protein Synthesis Inhibitors; D058928:Riboswitch; D013113:Spin Labels; D013752:Tetracycline