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

• Published in: Chembiochem : a European journal of chemical biology Vol. 15; no. 11; pp. 1627 - 1637
• Main Authors: Weigand, Julia E., Gottstein-Schmidtke, Sina R., Demolli, Shemsi, Groher, Florian, Duchardt-Ferner, Elke, Wöhnert, Jens, Suess, Beatrix
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 21.07.2014; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: aptamers; Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - metabolism; Binding Sites; Biological activity; Efficiency; Gene expression; Ligands; Models, Molecular; Mutation; Neomycin - metabolism; NMR spectroscopy; Riboswitch - genetics; riboswitches; RNA structures; synthetic biology; synthetic biology; RNA structures; riboswitches; NMR spectroscopy; aptamers; gene expression
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201402067

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. The structure–function relationship of a U‐turn motif in the terminal loop of a synthetic neomycin riboswitch has been investigated. The analysis revealed those nucleobases required to optimize riboswitch function at every single position. A detailed comparison of functional and inactive mutants revealed a relationship between aptamer dynamics and regulation efficiency.