Mechanism of post‐segregational killing: Sok antisense RNA interacts with Hok mRNA via its 5′‐end single‐stranded leader and competes with the 3′‐end of Hok mRNA for binding to the mok translational initiation region.

The hok/sok system of plasmid R1, which mediates plasmid stabilization by killing of plasmid‐free segregants, codes for two RNA species, Hok mRNA and Sok antisense RNA. The lethal expression of hok is inhibited post‐transcriptionally by the 67 nt Sok‐RNA. In this paper, we analyse the secondary structure of Sok‐RNA and the binding of Sok‐RNA to Hok mRNA in vitro. The reaction between the two RNAs leads to the formation of a complete duplex in which Sok‐RNA is hybridized over its entire length to Hok mRNA. The second‐order rate constant of duplex formation was determined to be approximately 1 × 10(5) M‐1s‐1. Mutations in the 5′‐end single‐stranded leader of Sok‐RNA severely reduced the binding rate to wt Hok mRNA, whereas loop mutations in Sok‐RNA had no such effect. The reduced binding rates were paralleled by abolished in vivo regulatory properties. These results suggest that, unlike in other well‐characterized antisense/target RNA systems, the initial recognition reaction between Sok‐RNA and Hok mRNA takes place between the single‐stranded 5′‐end of Sok‐RNA and the complementary region in Hok mRNA, without the involvement of an antisense loop in the initial binding step. Furthermore, the finding that Sok‐RNA competes with the 3′‐end of full‐length Hok mRNA for binding to the mok translational initiation region adds to the complexity of killer gene regulation.