Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT), The stable CopA-CopT complex is characterized by a four -way junction structure and a side-by-side alignment of two long intramolec ular helices. The significance of this structure for binding in vitro and c ontrol lit vivo was tested by mutations in both CopA and CopT, High rates o f stable complex formation in vitro and efficient inhibition in vivo requir ed initial loop-loop complexes to be rapidly converted to extended interact ions. These interactions involve asymmetric helix progression and melting o f the upper stems of both RNAs to promote the formation of two intermolecul ar helices. Data presented here delineate the boundaries of these helices a nd emphasize the need for unimpeded helix propagation. This process is dire ctional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hi erarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.