Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid

Abstract

The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene. To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage. Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein. The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY. We also isolated mutants that suppressed the rep57 phenotype. These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ. Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression. We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal. The positive and negative regulations of repZ expression were discussed. The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.