title: A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy.
abstract: The 5' untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II-IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg(2+)-induced switch between two alternative conformations: from 'open', elongated morphologies at 0-2 mM Mg(2+) concentration to a 'closed', comma-shaped conformation at 4-6 mM Mg(2+). This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4-6 mM Mg(2+) in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I-II)+(V-VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, (CIBERehd), Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: García-Sacristán
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Moreno
authors.affiliation: Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, (CIBERehd), Spain Laboratory of RNA Archaeology, Instituto de Parasitología y Biomedicina 'López-Neyra' (CSIC), Parque Tecnológico Ciencias de la Salud, Armilla, Granada 18016, Spain.
authors.forename: Ascensión
authors.initials: A
authors.lastname: Ariza-Mateos
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, Madrid 28049, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: López-Camacho
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Jáudenes
authors.affiliation: Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, Madrid 28049, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Vázquez
authors.affiliation: Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, (CIBERehd), Spain Laboratory of RNA Archaeology, Instituto de Parasitología y Biomedicina 'López-Neyra' (CSIC), Parque Tecnológico Ciencias de la Salud, Armilla, Granada 18016, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Gómez
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, Madrid 28049, Spain.
authors.forename: José Ángel
authors.initials: JÁ
authors.lastname: Martín-Gago
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, (CIBERehd), Spain cbriones@cab.inta-csic.es.
authors.forename: Carlos
authors.initials: C
authors.lastname: Briones
mesh_terms: D020121:5' Untranslated Regions; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D008274:Magnesium / Q000494:pharmacology*; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D059370:RNA Folding / Q000187:drug effects; D012367:RNA, Viral / Q000737:chemistry* / Q000648:ultrastructure; D012270:Ribosomes / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D008274:Magnesium