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Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.

Wunnicke, Dorith and Strohbach, Denise and Weigand, Julia E. and Appel, Bettina and Feresin, Emiliano and Suess, Beatrix and Müller, Sabine and 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), pp. 182-8. ISSN 1469-9001,
[Article]

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.

Item Type: Article
Erschienen: 2011
Creators: Wunnicke, Dorith and Strohbach, Denise and Weigand, Julia E. and Appel, Bettina and Feresin, Emiliano and Suess, Beatrix and Müller, Sabine and Steinhoff, Heinz-Jürgen
Title: Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.
Language: English
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.

Journal or Publication Title: RNA (New York, N.Y.)
Journal volume: 17
Number: 1
Divisions: 10 Department of Biology
10 Department of Biology > Synthetic Genetic Circuits
10 Department of Biology > RNA Biochemistry
Date Deposited: 22 Feb 2012 10:20
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