Control of ColE2 plasmid replication: negative regulation of the expression of the plasmid-specified initiator protein, Rep, at a posttranscriptional step.

The incA gene of ColE2 is involved in the copy number control and incompatibility. Two promoters were identified around the incA gene. Transcription of the mRNA for the essential plasmid-coded initiator protein (Rep) mainly starts at a site about 140 bp upstream of the initiation codon of the Rep protein. The second transcript (RNA I) of about 115 nucleotides with two stem-and-loop structures is entirely complementary to the 5' untranslated region of the Rep mRNA. By using translational and transcriptional fusions of the rep gene of ColE2 and the lacZ gene of Escherichia coli, the incA gene product was shown to regulate expression of the rep gene at a posttranscriptional step. The results also suggest that the target of the incA gene product is the 5' untranslated region of the Rep mRNA. Deletion analyses reported here show that a region(s) about 17 to 70 bp upstream of the initiation codon of the Rep protein and another region inside the coding frame are important for efficient production of the Rep protein. This suggests that some additional sequence elements other than the initiation codon and the Shine-Dalgarno region and/or a secondary structure of the Rep mRNA are required for efficient production of the Rep protein. These results show that RNA I is an antisense RNA for the Rep mRNA and imply that it might regulate expression of the rep gene at the initiation step of translation by sequestering such additional sequence elements and/or by disrupting RNA secondary structure. We propose that RNA I represents the incA gene product.