BULGED-OUT NUCLEOTIDES IN AN ANTISENSE RNA ARE REQUIRED FOR RAPID TARGET RNA-BINDING IN-VITRO AND INHIBITION IN-VIVO

Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated, Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA, Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions, Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo, The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression, A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex', We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA, The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.