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 x 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.
Thisted
T
T
Department of Molecular Biology, Odense University, Denmark.
Sørensen
N S
NS
Wagner
E G
EG
Gerdes
K
K
D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D011815:R Factors / Q000235:genetics*; D012313:RNA; D016372:RNA, Antisense / Q000378:metabolism; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D017384:Sequence Deletion
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; C087026:Sok antisense RNA, E coli; D012313:RNA