Structure of the three-way helical junction of the hepatitis C virus IRES element

Jonathan Ouellet, Sonya Melcher, Asif Iqbal, Yiliang Ding, David M. J. Lilley

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    Abstract

    The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS(4) junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS(1)HS(3) and 2HS(2)HS(2) junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg2+ ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.

    Original languageEnglish
    Pages (from-to)1597-1609
    Number of pages13
    JournalRNA: a Publication of the RNA Society
    Volume16
    Issue number8
    DOIs
    Publication statusPublished - Aug 2010

    Keywords

    • RNA structure
    • RNA junctions
    • translation
    • FRET
    • single-molecule fluorescence
    • INTERNAL RIBOSOME ENTRY
    • COMPARATIVE GEL-ELECTROPHORESIS
    • RESONANCE ENERGY-TRANSFER
    • 5' NONTRANSLATED REGIONS
    • DOUBLE-STRANDED DNA
    • HAMMERHEAD RIBOZYME
    • CRYSTAL-STRUCTURE
    • HAIRPIN RIBOZYME
    • TRANSLATION INITIATION
    • LYSINE RIBOSWITCH

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