MECHANISM OF BINDING OF THE ANTISENSE AND TARGET RNAS INVOLVED IN THEREGULATION 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 struc tured, antisense RNA (RNA I) to its complementary target in the RepA m RNA (RNA II) inhibits repA expression and thus regulates replication. Analyses of binding of RNA I to RNA II indicated that the reaction con sists of three major steps. The first step, initial kissing complex fo rmation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II. The second step is facilitated by i nterior loop structures in the upper stems of RNA I and RNA II and inv olves intrastrand melting and interstrand pairing of the upper stem re gions to form an extended kissing complex. This complex was shown to b e sufficient for inhibition of repA expression. The third step involve s stabilization of the extended kissing complex by pairing between com plementary 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 th e two molecules.