Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR
Autor(en): | Wunnicke, Dorith Strohbach, Denise Weigand, Julia E. Appel, Bettina Feresin, Emiliano Suess, Beatrix Mueller, Sabine Steinhoff, Heinz-Juergen |
Stichwörter: | APTAMERS; BINDING RIBOSWITCH; Biochemistry & Molecular Biology; DEER; DISTANCE MEASUREMENTS; DYNAMICS; ELECTRON-PARAMAGNETIC-RESONANCE; ENERGY-TRANSFER; ESR; NUCLEIC-ACIDS; RNA; RNA folding; SITE; site-directed spin labeling; SPECTROSCOPY; synthetic riboswitch; tetracycline aptamer | Erscheinungsdatum: | 2011 | Herausgeber: | COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT | Journal: | RNA | Volumen: | 17 | Ausgabe: | 1 | Startseite: | 182 | Seitenende: | 188 | Zusammenfassung: | 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. |
ISSN: | 13558382 | DOI: | 10.1261/rna.2222811 |
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