An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA‐CopT pairing in vitro showed that the dominant product of antisense RNA‐mRNA binding is not a full RNA duplex. We have studied here the structure of CopA‐CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop‐loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA‐CopT RNA complex. This stabilizer helix cannot form in CopI‐CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI‐CopT) and the stable complex (CopA‐CopT) are discussed.