Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure

Abstract

Efficient gene control by antisense RNA requires rapid bi-molecular interaction with a cognate target RNA. A comparative analysis revealed that a YUNR motif (Y=pyrimidine, R=purine) is ubiquitous in RNA recognition loops in antisense RNA-regulated gene systems. The (Y)UNR sequence motif specifies two intraloop hydrogen bonds forming U-turn structures in many anticodon-loops and all T-loops of tRNAs, the hammerhead ribozyme and in other conserved RNA loops. This structure creates a sharp bend in the RNA phosphate-backbone and presents the following three to four bases in a solvent-exposed, stacked configuration providing a scaffold for rapid interaction with complementary RNA. Sok antisense RNA from plasmid R1 inhibits translation of the hok mRNA by preventing ribosome entry at the mok Shine & Dalgarno element. The 5′ single-stranded region of Sok-RNA recognizes a loop in the hok mRNA. We show here, that the initial pairing between Sok antisense RNA and its target in hok mRNA occurs with an observed second-order rate-constant of 2×10 6 M −1 s −1. Mutations that eliminate the YUNR motif in the target loop of hok mRNA resulted in reduced antisense RNA pairing kinetics, whereas mutations maintaining the YUNR motif were silent. In addition, RNA phosphate-backbone accessibility probing by ethylnitrosourea was consistent with a U-turn structure formation promoted by the YUNR motif. Since the YUNR U-turn motif is present in the recognition units of many antisense/target pairs, the motif is likely to be a generally employed enhancer of RNA pairing rates. This suggestion is consistent with the re-interpretation of the mutational analyses of several antisense control systems including RNAI/RNAII of ColE1, CopA/CopT of R1 and RNA-IN/RNA-OUT of IS 10.