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J. Biol. Chem., Vol. 278, Issue 37, 35558-35563, September 12, 2003

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Loop Swapping in an Antisense RNA/Target RNA Pair Changes Directionality of Helix Progression^*

Jacoba G. Slagter-Jäger and E. Gerhart H. Wagner 

From the Institute of Cell and Molecular Biology, Department of Microbiology, Biomedical Center, Uppsala University, Box 596, Husargatan 3, S-75124 Uppsala, Sweden

The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or -turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.

Received for publication, May 9, 2003 , and in revised form, June 18, 2003.

^* This work was supported in part by the Swedish Science Research Council, the Foundation for Strategic Research, and the Wallenberg Consortium North. 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.

To whom correspondence should be addressed: Dept. of Microbiology, Institute of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, Husargatan 3, S-75124 Uppsala, Sweden. Tel.: 46-18-4714866; Fax: 46-18-5309396; E-mail: Gerhart.Wagner{at}icm.uu.se.

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