MECHANISM OF POST-SEGREGATIONAL KILLING - SOK ANTISENSE RNA INTERACTSWITH HOK MESSENGER-RNA VIA ITS 5'-END SINGLE-STRANDED LEADER AND COMPETES WITH THE 3'-END OF HOK MESSENGER-RNA 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 analys e 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 formati on of a complete duplex in which Sok-RNA is hybridized over its entire length to Hok mRNA. The second-order rate constant of duplex formatio n was determined to be similar to 1 x 10(5) M(-1)s(-1). Mutations in t he 5'-end single-stranded leader of Sok-RNA severely reduced the bindi ng 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 we ll-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, w ithout the involvement of an antisense loop in the initial binding ste p. Furthermore, the finding that Sok-RNA competes with the 3'-end of f ull-length Hok mRNA for binding to the mok translational initiation re gion adds to the complexity of killer gene regulation.