THE REPLICATION OF AN INCL M PLASMID TS SUBJECT TO ANTISENSE CONTROL/

A 2,385-bp sequence that contains the information for the autonomous r eplication of the IncL/M plasmid pMU604 was characterized. Genetic ana lyses revealed that the replicon specifies at least four structural ge nes, designated repA, repB, repC and rnaI. The repA gene encodes a pro tein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB a nd repC genes are unclear; however, mutations in the start codon of re pB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnaI gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incom patibility phenotype, thus implicating its role as the main copy numbe r determinant. RNAI exerts its effect in trans to repress the expressi on of repA at the posttranscriptional level. Furthermore, two compleme ntary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences w as shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational couplin g and pseudoknot formation is proposed.