Control of ColE2 DNA replication: in vitro binding of the antisense RNA to the Rep mRNA

Expression of the Rep protein of colicin E2 plasmid is negatively controlled at a post-transcriptional step by a plasmid-coded RNA (RNAI). RNAI is complementary to the 5' nontranslated region of the Rep mRNA and can be folded into two stem-loop structures. Several cop mutations that increased the copy number of the plasmid have been mapped in the RNAI coding region. Here we demonstrated that RNAI and the Rep mRNA rapidly form a stable complex under near physiological conditions. The cop mutations drastically reduced the binding rate. The reduction correlated to the in vivo phenotypes of the cop mutant plasmids, indicating that binding of RNAI to the Rep mRNA is involved in the regulation of the Rep protein expression. Binding properties of RNAI and the Rep mRNA with the cop mutations and those with various extents of deletion suggested that the initial interaction of the two RNAs occurs between the loop portions of the larger of the two stem-loop structures. RNAI does not completely sequester the putative S/D region, even when the two RNAs form a complete duplex. Possible mechanisms of the inhibition of the rep gene expression by binding of RNAI to the Rep mRNA under such a situation were discussed.