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Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch.

Weigand, Julia E. and Gottstein-Schmidtke, Sina R. and Demolli, Shemsi and Groher, Florian and Duchardt-Ferner, Elke and Wöhnert, Jens and Suess, Beatrix (2014):
Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch.
In: Chembiochem : a European journal of chemical biology, pp. 1626-1636, 15, (11), ISSN 1439-7633, [Article]

Abstract

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA-ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence-activity relationships that could not initially be explained on the basis of the structure of the aptamer-ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.

Item Type: Article
Erschienen: 2014
Creators: Weigand, Julia E. and Gottstein-Schmidtke, Sina R. and Demolli, Shemsi and Groher, Florian and Duchardt-Ferner, Elke and Wöhnert, Jens and Suess, Beatrix
Title: Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch.
Language: English
Abstract:

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA-ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence-activity relationships that could not initially be explained on the basis of the structure of the aptamer-ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.

Journal or Publication Title: Chembiochem : a European journal of chemical biology
Volume: 15
Number: 11
Divisions: 10 Department of Biology
10 Department of Biology > Synthetic Genetic Circuits
Date Deposited: 01 Jul 2014 11:09
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