Localized domains of DNA supercoiling: topological coupling between promoters.
According to the twin-supercoiled domain model, a local domain with a high level of DNA supercoiling can be generated between two divergently transcribed promoters. We have tested this model directly using the rate of DNA photo-cross-linking by 4,5', 8-trimethylpsoralen to measure local regions of unconstrained supercoiling in vivo. We demonstrate that, in plasmids, a localized domain of highly supercoiled DNA is generated upstream of the tetA promoter. The level of supercoiling of this domain depends on the activity of the tetA promoter and on the extent of translation of the tetA gene product. The highest level of supercoiling within this localized domain was observed in a topA strain, although the generation of a localized supercoiling domain could also be detected in a topA+ background, consistent with potential physiological significance. The level of supercoiling in this localized domain correlated with activation of the supercoiling-sensitive leu-500 promoter located upstream of tetA. These data provide a direct demonstration that localized domains of increased negative supercoiling can be generated upstream of an actively transcribed promoter, and that this can result in supercoiling-mediated transcriptional coupling between two promoters.
Mojica
F J
FJ
Nuffield Department of Clinical Biochemistry, Imperial Cancer Research Fund Laboratories, University of Oxford, John Radcliffe Hospital, UK.
Higgins
C F
CF
D017920:Antiporters / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000187:drug effects*; D004278:DNA, Superhelical; D007364:Intercalating Agents / Q000494:pharmacology; D017319:Photosensitizing Agents / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014307:Trioxsalen / Q000494:pharmacology
D017920:Antiporters; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D007364:Intercalating Agents; D017319:Photosensitizing Agents; C079051:tetA protein, Bacteria; D014307:Trioxsalen