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.
Highlights
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
Reversal of the tetA Gene of pLEU500Tc—In previous studies we showed that the activation of the leu-500 promoter on the plasmid pLEU500Tc in topA S. typhimurium was dependent on the function of the adjacent tetracycline resistance gene tetA [10]
Summary
The leu-500 promoter is inactivated by a mutation in the ؊10 region but can be activated in topA Escherichia coli and Salmonella strains. 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. Using such plasmids we could achieve topA-dependent activation of the promoter in either Salmonella [10] or Escherichia coli [14] This implied a key role for the tetA gene, and a number of studies have indicated that the coupled transcription, translation, and membrane insertion of the tetA gene product are essential for efficient oversupercoiling of plasmids in topA eubacterial cells [13, 15,16,17] due to the anchorage of the transcribing RNA polymerase to the membrane. A point of anchorage (such as the insertion of the nascent TetA polypeptide into the membrane) should provide a barrier to the diffusion of supercoiling around the plasmid and might increase local levels of DNA supercoiling
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