Twin domains of positive and negative supercoiling are thought to form in DNA molecules whenever free rotation of a transcription complex around the DNA helix is impeded. Evidence for these domains has come from findings with Escherichia coli strains that are deficient in DNA topoisomerase I (top mutants) or that have been treated with DNA gyrase inhibitors. Plasmid pBR322 is hghly supercoiled in these strains, whereas some of its deletion derivatives are not. The studies of pBR322 derivatives presented here show that high negative supercoiling in top strains requires translation as well as transcription of the first 98 codons of the tet gene and does not require the divergently transcribed amp gene. The N-terminal region of the TetA protein is thought to insert into the inner membrane. Our results favor models in which supercoiling domains are created when DNA segments are anchored to a large cellular structure via coupled transcription, translation, and membrane insertion of a nascent protein.