Exploring TAR-RNA aptamer loop-loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance.
Résumé
In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5' untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducong?. and Toulm?J (1999) RNA, 5:1605-1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson-Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1 degrees , thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2' hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2'-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability.
Domaines
Biochimie, Biologie Moléculaire
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LebarsHAL.pdf (766.27 Ko)
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SI.pdf (211.31 Ko)
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