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 domi nant product of antisense RNA-mRNA binding is not a full RNA duplex. We hav e studied here the structure of CopA-CopT complex, combining chemical and e nzymatic probing and computer graphic modeling. CopI, a truncated derivativ e of CopA unable to bind CopT stably, was also analyzed, We show here that after initial loop-loop interaction (kissing), helix propagation resulted i n an extended kissing complex that involves the formation of two intermolec ular helices, By introducing mutations (base-pair inversions) into the uppe r stem regions of CopA and CopT, the boundaries of the two newly formed int ermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmet rical X-shaped conformation formed by two helical domains, each one generat ed by coaxial stacking of two helices, This structure motif induces a side- by-side alignment of two long intramolecular helices that, in turn, facilit ates the formation of an additional intermolecular helix that greatly stabi lizes the inhibitory CopA-CopT RNA complex, This stabilizer helix cannot fo rm in CopI-CopT complexes due to absence of the sequences involved, The fun ctional significance of the three-dimensional models of the extended kissin g complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.