title: The NMR structure of the II-III-VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure.
abstract: As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III-IV-V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II-III-VI three-way junction and its adjoining stems. In addition, we localize Mg(2+)-binding sites within this structure using Mn(2+)-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg(2+) ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge-bulge interaction and Mg(2+) ions are critical for the stable folding of the II-III-VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II-III-VI junction that does not contain the II-VI bulge-bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme.
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneau
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Girard
authors.affiliation: Département d'Informatique et de Recherche Opérationnelle et Institut de Recherche en Immunologie et en Cancérologie, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lemieux
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.
authors.forename: Pascale
authors.initials: P
authors.lastname: Legault
mesh_terms: D020029:Base Pairing; D001665:Binding Sites; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009491:Neurospora / Q000737:chemistry / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry* / Q000378:metabolism
chemical_list: D016337:RNA, Catalytic; D012331:RNA, Fungal; D004722:Endoribonucleases; C438195:varkud satellite ribozyme; D008274:Magnesium
keywords: NMR structure; RNA three-way junction; Varkud satellite ribozyme; magnesium ions; remote tertiary interaction