REPLICATION CONTROL OF PLASMID-R1 - REPA SYNTHESIS IS REGULATED BY COPA RNA THROUGH INHIBITION OF LEADER PEPTIDE TRANSLATION

The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded. A model accounting for the involvement of tap in copy number regulation of plasmid R1 is presented, and its implications are discussed in relation to studies of the IncF-alpha plasmid ColIb-P9, in which a similar leader peptide has been described (Hama et al., 1990; Asano et al., 1991a,b).