ACTIVATION OF THE LEU-500 PROMOTER - A TOPOLOGICAL DOMAIN GENERATED BY DIVERGENT TRANSCRIPTION IN A PLASMID

The Salmonella typhimurium leu-500 promoter is active only in topA strains. In an earlier study (Chen, D., Bowater, R., Dorman, C., & Lilley, D. M. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8784-8788), we showed that the activity of this promoter on a circular plasmid is a function of the transcription and translation of an adjacent tetA gene, and we suggested that the effect arises because of increased local negative superhelix density due to transcription (Liu, L. F., & Wang, J. C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7024-7027) initiated at the tetA promoter. In this study we show that translation of the 5' (N-terminal) section of tetA is required for activity of the leu-500 promoter, consistent with a requirement for membrane association of TetA. We have also shown the importance of a second transcription unit, the ampicillin resistance gene bla, in the activation of the leu-500 promoter. Thus the activity of the leu-500 promoter was reduced by partial deletion or premature termination of bla and was increased when the transcription of bla was boosted by the insertion of the stronger tac promoter. However, even in the latter situation the role of the tetA gene is dominant, and deletion of the tetA gene reduced activity of the leu-500 promoter to very low levels. These results suggest the existence of a topological domain defined by the divergent bla and tetA transcription units. Membrane insertion at tetA is essential to provide an anchorage point. Insertion of random DNA sequences within the bla-to-tetA domain resulted in a reduction in initiation of transcription at the leu-500 promoter, whereas insertion outside the domain had almost no effect. These observations are consistent with activation of the leu-500 promoter by negative supercoiling in the bla-to-tetA domain, the steady-state superhelix density of which is a function of the relative rates of induction by transcription and relaxation, and the length of DNA between the divergent genes.