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A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

AuthorsGarcía-Sacristán, Ana CSIC ORCID; Moreno Molina, Miguel CSIC ORCID CVN; Ariza-Mateos, Ascensión CSIC; López-Camacho, Elena CSIC; Jáudenes, Rosa M.; Vázquez, Luis CSIC ORCID ; Gómez, Jordi; Martín-Gago, José A. CSIC ORCID ; Briones, Carlos CSIC ORCID
Issue DateDec-2014
PublisherOxford University Press
CitationNucleic Acids Research
AbstractThe 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, Mg2+-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg2+. 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 Mg2+ 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.
DescriptionSupplementary data.
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