Wunnicke, Dorith ; Strohbach, Denise ; Weigand, Julia E. ; Appel, Bettina ; Feresin, Emiliano ; Suess, Beatrix ; Müller, Sabine ; Steinhoff, Heinz-Jürgen (2011)
Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.
In: RNA (New York, N.Y.), 17 (1)
Artikel, Bibliographie

Kurzbeschreibung (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.

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