title: Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo.
abstract: 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.
authors.affiliation: Department of Microbiology, Uppsala University, Sweden.
authors.forename: T A
authors.initials: TA
authors.lastname: Hjalt
authors.forename: E G
authors.initials: EG
authors.lastname: Wagner
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D003086:Bacteriocin Plasmids / Q000235:genetics; D001665:Binding Sites; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D011506:Proteins; D011815:R Factors / Q000235:genetics*; D016372:RNA, Antisense / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015534:Trans-Activators
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011506:Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases