Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR
| Autor: | Denise Strohbach, Dorith Wunnicke, Julia E. Weigand, Bettina Appel, Sabine Müller, Beatrix Suess, Heinz-Jürgen Steinhoff, Emiliano Feresin |
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| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Riboswitch
Models Molecular Protein Synthesis Inhibitors Stereochemistry Pulsed EPR Aptamer Electron Spin Resonance Spectroscopy RNA Site-directed spin labeling Biology Aptamers Nucleotide Tetracycline Ligand (biochemistry) Ligands Article Biochemistry Gene expression Nucleic Acid Conformation Thermodynamics Spin Labels Spin label Molecular Biology |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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