Large-scale effects of transcriptional DNA supercoiling in vivo

The scale of negative DNA supercoiling generated by transcription in Top(+) Escherichia coli cells was assessed from the efficiency of cruciform formation upstream of a regulated promoter. An increase in negative supercoiling upon promoter induction led to cruciform formation, which was quantitatively measured by chemical probing of intracellular DNA. By placing a cruciform-forming sequence at varying distances from the promoter, we found that the half-dissociation length of transcription supercoiling wave is approximate to 800 bp. This is the first proof that transcription can affect DNA structure on such a remarkably large scale in vivo. Moreover, cooperative binding of the cI repressor to the upstream promoter DNA did not preclude efficient diffusion of transcriptional supercoiling. Finally, our plasmids appeared to contain discrete domains of DNA supercoiling, defined by the features and relative orientation of different promoters. (C) 1999 Academic Press.