Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome.

Abstract

The notion that transcription can generate supercoils in the DNA template largely stems from work with small circular plasmids. In the present work, we tested this model in the bacterial chromosome using a supercoiling-sensitive promoter as a functional sensor of superhelicity changes. The leu-500 promoter of Salmonella typhimurium is a mutant and inactive variant of the leucine operon promoter that regains activity if negative DNA supercoiling rises above normal levels, typically as a result of mutations affecting DNA topoisomerase I (topA mutants). Activation of the leu-500 promoter was analysed in topA mutant cells harbouring transcriptionally inducible tet or cat gene cassettes inserted in the region upstream from the leu operon. Some insertions inhibited leu-500 promoter activation in the absence of inducer. This effect is dramatic in the interval between 1.7 kb and 0.6 kb from the leu operon, suggesting that the insertions physically interfere with the mechanism responsible for activation. Superimposed on these effects, transcription of the inserted gene stimulated or inhibited leu-500 promoter activity depending on whether this gene was oriented divergently from the leu operon or in the same direction respectively. Interestingly, transcription-mediated inhibition of leu-500 promoter was observed with inserts as far as 5 kb from the leu operon, and it could be relieved by the introduction of a strong gyrase site between the inserted element and the leu-500 promoter. These results are consistent with the idea that transcriptionally generated positive and negative supercoils can diffuse along chromosomal DNA and, depending on their topological sign, elicit opposite responses from the leu-500 promoter.