Activation of the leu-500 promoter by a reversed polarity tetA gene. Response to global plasmid supercoiling

Abstract

The leu-500 promoter is inactivated by a mutation in the -10 region but can be activated in topA Escherichia coli and Salmonella strains. We have found that the tetA gene plays a vital role in the topA-dependent activation of a plasmid-borne leu-500 promoter. In previous studies, the leu-500 promoter and tetA gene have been arranged divergently. In this study we have reversed the polarity of the tetA gene, thus locating the leu-500 promoter at the 3' end of tetA. Despite being formally located in the downstream region of tetA, the leu-500 promoter is equally well activated in a topA strain in this environment, even though it is 1.6 kilobase pairs away from the promoter of the reversed tetA gene. Activation of the leu-500 promoter depends on transcription and translation of tetA but is largely insensitive to the function of other transcription units on the plasmid. These results require a change in viewpoint of the role of tetA, from local to global supercoiling. We conclude that transcription of the tetA gene is the main generator of transcription-induced supercoiling that activates the leu-500 promoter. Unbalanced relaxation of this supercoiling leads to a net increase in the negative linking difference of the plasmid globally, and there is a linear correlation between the change in global plasmid topology and the activation of the leu-500 promoter. Thus the leu-500 promoter appears to respond to the negative supercoiling of the plasmid overall.