title: Screening for engineered neomycin riboswitches that control translation initiation.
abstract: Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality.
authors.affiliation: Institut für Molekulare Biowissenschaften, Johann-Wolfgang-Goethe-Universität Frankfurt, 60438 Frankfurt am Main, Germany.
authors.forename: Julia E
authors.initials: JE
authors.lastname: Weigand
authors.forename: Martin
authors.initials: M
authors.lastname: Sanchez
authors.forename: Ewald-Bernd
authors.initials: EB
authors.lastname: Gunnesch
authors.forename: Sabrina
authors.initials: S
authors.lastname: Zeiher
authors.forename: Renee
authors.initials: R
authors.lastname: Schroeder
authors.forename: Beatrix
authors.initials: B
authors.lastname: Suess
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology*; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship
chemical_list: D012335:RNA, Ribosomal; D009355:Neomycin