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J. Biol. Chem., Vol. 280, Issue 48, 40112-40121, December 2, 2005

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Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site^*

Serena Bernacchi12, Eric Ennifar1, Katalin Tóth, Philippe Walter, Jörg Langowski, and Philippe Dumas3

From the Institut de Biologie Moléculaire et Cellulaire, UPR 9002 du CNRS Conventionnée à l'Université Louis Pasteur Strasbourg, 15 Rue René Descartes, F-67084 Strasbourg Cedex, France and German Cancer Research Center, Division of Biophysics of Macromolecules, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany

We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 µM in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 µM in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.

Received for publication, March 23, 2005 , and in revised form, August 19, 2005.

^* This work was supported by the Agence Nationale de Recherche sur le SIDA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1-S4.

¹ Both authors contributed equally to this work.

² A German Cancer Research Center (Deutsches KrebsForschungsZentrum) fellow.

³ To whom correspondence should be addressed. Tel.: 33-388-417-002; Fax: 33-388-602-218; E-mail: p.dumas{at}ibmc.u-strasbg.fr.

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