The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repAΔC143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repAΔC143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42°C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repAΔC143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37°C. At 42°C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.
All Science Journal Classification (ASJC) codes
- Molecular Biology