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Volume 272, Number 19, Issue of May 9, 1997 pp. 12508-12512
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Antisense RNA Control of Plasmid R1 Replication
THE DOMINANT PRODUCT OF THE ANTISENSE RNA-mRNA BINDING IS NOT A FULL RNA DUPLEX

(Received for publication, January 31, 1997)

From the Department of Microbiology, Biomedical Center, Uppsala University, Box 581 S-751 23 Uppsala, Sweden, ^§ Department of Microbiology, Uppsala Genetic Center, SLU (Swedish University of Agricultural Sciences), Box 7025, Genetikvägen 5, S-75007 Uppsala, Sweden, and ^¶ UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue R. Descartes, Strasbourg cedex, France

The replication frequency of plasmid R1 is controlled by an antisense RNA (CopA) that binds to its target site (CopT) in the leader region of repA mRNA and inhibits the synthesis of the replication initiator protein RepA. Previous studies on CopA-CopT pairing in vitro revealed the existence of a primary loop-loop interaction (kissing complex) that is subsequently converted to an almost irreversible duplex. However, the structure of more stable binding intermediates that lead to the formation of a complete duplex was speculative. Here, we investigated the interaction between CopA and CopT by using Pb(II)-induced cleavages. The kissing complex was studied using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT. Furthermore, RNase III, which is known to process the CopA-CopT complex in vivo, was used to detect the existence of a full duplex. Our data indicate that the formation of a full CopA-CopT duplex appears to be a very slow process in vitro. Unexpectedly, we found that the loop-loop interaction persists in the predominant CopA-CopT complex and is stabilized by intermolecular base pairing involving the 5-proximal 30 nucleotides of CopA and the complementary region of CopT. This almost irreversible complex suffices to inhibit ribosome binding at the tap ribosome binding site and may be the inhibitory complex in vivo.

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