MECHANISM OF BINDING OF THE ANTISENSE AND TARGET RNAS INVOLVED IN THE REGULATION OF INCB PLASMID REPLICATION

The replication frequency of the IncB miniplasmid pMU720 is dependent upon the expression of the repA gene. Binding of a small, highly structured, antisense RNA (RNA I) to its complementary target in the RepA mRNA (RNA II) inhibits repA expression and thus regulates replication. Analyses of binding of RNA I to RNA II indicated that the reaction consists of three major steps. The first step, initial kissing complex formation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II. The second step is facilitated by interior loop structures in the upper stems of RNA I and RNA II and involves intrastrand melting and interstrand pairing of the upper stem regions to form an extended kissing complex. This complex was shown to be sufficient for inhibition of repA expression. The third step involves stabilization of the extended kissing complex by pairing between complementary single-stranded tail regions of RNA I and RNA II. Thus, the final product of RNA I-RNA II binding is not a full duplex between the two molecules.