HYPER-NEGATIVE TEMPLATE DNA SUPERCOILING DURING TRANSCRIPTION OF THE TETRACYCLINE-RESISTANCE GENE IN TOPA MUTANTS IS LARGELY CONSTRAINED IN-VIVO

The excess linking deficit of plasmid DNA from topoisomerase I-defecti ve bacteria (topA mutants) results mainly from transcription and is co mmonly ascribed to unbalanced relaxation of transcription-induced twin -supercoiled domains. This defect is aggravated in genes for membrane- binding proteins (such as the tet gene) where anchoring of the transcr iption complex to the bacterial membrane is thought to enhance twin-do main partitioning. Thus, it is often assumed that the 'hyper-negative' linking difference of plasmid DNA from topA mutants reflects unconstr ained, hyper-negative DNA supercoiling inside the cell. We tested the validity of this assumption in the present study. A DNA sequence that undergoes a gradual B to Z transition under increasing negative superh elical tension was used as a sensor of unconstrained negative supercoi ling. Z-DNA formation was probed at a site upstream from the inducible pTac promoter fused either to the tet gene or to the gene for cytosol ic chloramphenicol acetyl transferase (cat), Although plasmid DNA link ing deficit increased more extensively in topA mutants following tet a ctivation than following cat activation, no significant differences we re observed in the extents to which the B to Z DNA transition is stimu lated in the two cases. We infer that the excess linking deficit of th e tet-containing plasmid DNA reflects constrained negative DNA superco iling inside the cell.