Abstract
Expression of the lacZ gene from the supercoiling-sensitive leu-500 promoter on a plasmid in topA mutant cells was stimulated by activating a divergently oriented Tac promoter, 400 bp upstream from leu-500. The stimulation was approximately threefold regardless of whether the Tac promoter drove the expression of the tet gene, whose product is membrane bound, or of the cat gene, whose product is cytosolic. Putting a second copy of the Tac promoter downstream from lacZ, approximately 3,000 bp from leu-500 in the same orientation as the latter, resulted in 30-fold increase in lacZ expression upon isopropyl-beta-D-thiogalactopyranoside induction. Again, these effects were independent of the nature of the gene upstream from leu-500 (tet or cat). With both tet- and cat-harboring constructs, activation of the two Tac promoter copies caused plasmid DNA to become hypernegatively supercoiled in topA mutant cells. Thus, neither leu-500 activation nor hypernegative plasmid DNA supercoiling appears to require membrane anchoring of DNA in this system. Replacing the downstream copy of Tac with a constitutive promoter resulted in high-level lacZ expression even when the upstream copy was repressed. Under these conditions, no hypernegative DNA supercoiling was observed, indicating that the activity of plasmid-borne leu-500 in topA mutant cells does not necessarily correlate with the linking deficit of plasmid DNA. The response of the leu-500-lacZ fusion to downstream transcription provides a sensitive assay for transcriptional supercoiling in bacteria.
Highlights
Translocation of RNA polymerase along the DNA template during transcription generates positive and negative supercoils [22, 41, 45]
Plasmids were introduced into two isogenic strains of S. typhimurium, a wild-type strain and a topA mutant, both expressing the E. coli lacIq repressor gene from an FЈ factor
Few hypernegatively supercoiled forms were produced, as judged from the weakness of the front band. Ascribing this observation to the merging and mutual annihilation of positive and negative supercoils, we speculated that the Tac-mediated stimulation of leu-500 in topA mutant cells might be boosted by the introduction of a sequence preventing supercoil diffusion downstream from lacZ
Summary
Translocation of RNA polymerase along the DNA template during transcription generates positive and negative supercoils [22, 41, 45]. Chen et al showed that a plasmid-borne leu-500 could become active in topA mutant cells when positioned near a divergently oriented tetA gene, provided that tetA transcription was coupled to translation and membrane anchoring of the nascent Tet polypeptide [7, 8]. Our working hypothesis was that activation of a leu500–lacZ fusion in topA mutant cells could be boosted upon introduction of a sequence preventing the diffusion of negative supercoils downstream from lacZ.
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