Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex

In several groups of bacterial plasmids, antisense RNAs regulate copy numbe r through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpai red residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topo logy facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged res idues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modi fication interference, and molecular modeling of bulged-less mutant CopA-Co pT complexes suggests that, subsequent to loop-loop contact, helix propagat ion is prevented. Instead, a fully base paired loop-loop interaction is for med, inducing a continuous stacking of three helices. Consequently, the sta bilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alon e failed to prevent ribosome binding at its loading site and, thus, inhibit ion of RepA translation was alleviated.