title: Building a stable RNA U-turn with a protonated cytidine.
abstract: The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.
authors.affiliation: Institute of Molecular Biosciences, Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany.
authors.forename: Sina R
authors.initials: SR
authors.lastname: Gottstein-Schmidtke
authors.affiliation: Institute of Molecular Biosciences, Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Duchardt-Ferner
authors.affiliation: Department of Biology, Technical University Darmstadt, 64287 Darmstadt, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Groher
authors.affiliation: Department of Biology, Technical University Darmstadt, 64287 Darmstadt, Germany.
authors.forename: Julia E
authors.initials: JE
authors.lastname: Weigand
authors.affiliation: Institute for Biophysical Chemistry, Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gottstein
authors.affiliation: Department of Biology, Technical University Darmstadt, 64287 Darmstadt, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Suess
authors.affiliation: Institute of Molecular Biosciences, Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Wöhnert
mesh_terms: D001483:Base Sequence; D003562:Cytidine; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D010710:Phosphates / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D058928:Riboswitch; D014529:Uridine
chemical_list: D010710:Phosphates; D058928:Riboswitch; D003562:Cytidine; D012313:RNA; D014529:Uridine
keywords: NMR; RNA structure; U-turn; hydrogen bond; protonated cytidine