Positive and negative regulations of plasmid CoLIb-P9 repZ gene expression at the translational level

Expression of the repZ gene involved in DNA replication of the ColIb-P9 plasmid depends on translation of a transcribed repZ leader sequence (repY) and is negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. To further understand the regulatory loop of repZ expression, we isolated and characterized replication-defective ColIb-P9 mutants that affected the level of repZ expression. Here we report that mutations occurring in two complementary sequences, one (5'GGCG3') in the inc region and one in the repY region, reduce the level of repZ expression without affecting transcription. The mutations in one complementary sequence were suppressed by compensatory base changes in the other sequence, restoring the ability of repZ expression. These results indicated that interaction by base pairing between the two complementary sequences of RepZ mRNA was essential for repZ translation. The two sequences, separated by 107 bases from each other, have a potential to form a novel pseudoknot in the RepZ mRNA leader. We also found that some mutations in the 5'GGCG3' sequence altered the specificity of Inc RNA, thereby reducing significantly its regulatory activity. Thus, this single specific sequence is involved in both positive and negative regulations for repZ expression. Possible regulatory mechanisms of repZ expression are discussed.