title: Sequence elements distal to the ligand binding pocket modulate the efficiency of a synthetic riboswitch.
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.
authors: Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany).
authors: Julia E
authors: JE
authors: Weigand
authors: Sina R
authors: SR
authors: Gottstein-Schmidtke
authors: Shemsi
authors: S
authors: Demolli
authors: Florian
authors: F
authors: Groher
authors: Elke
authors: E
authors: Duchardt-Ferner
authors: Jens
authors: J
authors: Wöhnert
authors: Beatrix
authors: B
authors: Suess
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D009355:Neomycin / Q000378:metabolism; D058928:Riboswitch / Q000235:genetics*
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D058928:Riboswitch; D009355:Neomycin
keywords: NMR spectroscopy; RNA structures; aptamers; gene expression; riboswitches; synthetic biology