Role of CopA to Regulate repABC Gene Expression on the Transcriptional Level

Abstract

Since replication of plasmids must be strictly controlled, plasmids that generally perform rolling circle replication generally maintain a constant copy number by strictly controlling the replication initiator Rep at the transcriptional and translational levels. Plasmid pJB01 contains three orfs (copA, repB, repC or repABC) consisting of a single operon. From analysis of amino acid sequence, pJB01 CopA was homologous to the Cops, as a copy number control protein, of other plasmids. When compared with a CopG of pMV158, CopA seems to form the RHH (ribbon-helix-helix) known as a motif of generalized repressor of plasmids. The result of gel mobility shift assay (EMSA) revealed that the purified fusion CopA protein binds to the operator region of the repABC operon. To examine the functional role of CopA on transcriptional level, 3 point mutants were constructed in coding frame of copA such as CopA R16M, K26R and E50V. The repABC mRNA levels of CopA R16M, K26R and E50V mutants increased 1.84, 1.78 and 2.86 folds more than that of CopA wt, respectively. Furthermore, copy numbers owing to mutations in three copA genes also increased 1.86, 1.68 and 2.89 folds more than that of copA wt, respectively. These results suggest that CopA is the transcriptional repressor, and lowers the copy number of pJB01 by reducing repABC mRNA and then RepB, as a replication initiator.