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

Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee DD1 5EH, United Kingdom.
RNA (Impact Factor: 4.94). 08/2010; 16(8):1597-609. DOI: 10.1261/rna.2158410
Source: PubMed

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 Mg(2+) 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.

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    • "The coaxial helical stacking status of each threeway junction in Dataset2 is described as one of three possibilities: H 1 H 2, H 2 H 3, H 1 H 3. Thus, every three-way junction in Dataset2 contains a coaxial stacking pattern. In the RNA literature, most research efforts have been focused on three-way and four-way junctions[6,15,606162partly due to the fact that higher-order junctions are rare. In particular, three-way junctions are the most abundant type of junctions, accounting for over 50% of the available crystal data. "
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    • "We have used our standard nomenclature for the nucleotides of k-turns (13), beginning with the G1b and A1n positions, insofar as possible. In the global structure of the junction, the T helix is coaxially aligned with the C helix, making this formally a Lex junction (24). Both G•A pairs are trans sugar (G)•Hoogsteen (A) pairs just as found in a standard k-turn structure, although the G1b•A1n pair is less buckled than is normally observed in a standard k-turn. "
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