Nucleic Acids Research Advance Access originally published online on November 7, 2008
Nucleic Acids Research 2008 36(22):7146-7156; doi:10.1093/nar/gkn831
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Nucleic Acids Research, 2008, Vol. 36, No. 22 7146-7156
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Structural Biology |
Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance
1CNRS-Université Bordeaux 1-ENITAB, UMR 5248 CBMN, Institut Européen de Chimie et Biologie, Pessac, F-33607, 2Synchrotron SOLEIL, L'Orme des Merisiers, Saint-Aubin, B.P. 48, 91192 Gif-sur-Yvette Cedex, 3INSERM U869, Institut Européen de Chimie et Biologie, Pessac, F-33607 and 4Université de Bordeaux, Bordeaux, F-33076, France
*To whom correspondence should be addressed. Tel: +33 5 40 00 30 63; Fax: +33 5 40 00 30 68; Email: s.fribourg{at}iecb.u-bordeaux.fr
Correspondence may also be addressed to Carmelo Di Primo. Tel: +33 5 40 00 30 50; Fax: +33 5 40 00 30 04; Email: c.diprimo{at}iecb.u-bordeaux.fr
Received August 11, 2008. Revised October 13, 2008. Accepted October 14, 2008.
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 [Ducongé F. and Toulmé JJ (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°, 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.
Present addresses: Isabelle Lebars, CNRS-Université Louis Pasteur, UMR 7104 IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries BP 10142, F-67404 Illkirch Cedex France
Noël Pinaud, CESAMO, Université de Bordeaux, 351 cours de la Libération, Talence, F-33405 cedex, France