Transcription-induced hypersupercoiling of plasmid DNA.

Transcription can induce high levels of negative supercoiling into plasmid DNA under some circumstances. This is especially true when the plasmid carries a functional tetracycline-resistance gene tetA, and is borne in a topA strain of Escherichia coli or Salmonella typhimurium. An important mechanism in transcription-induced supercoiling is believed to be the twin supercoiled-domain effect resulting from hindered rotation of the transcriptional complex, and this is very much more efficient where there is coupled transcription, translation and membrane insertion of the gene product. However, we have noted that strong promoters inserted into tetA-carrying plasmids can greatly increase the fraction of hypersupercoiled DNA. We show here that this effect is clearly present when the inserted promoter transcribes a very short segment of DNA (down to transcript lengths of approximately 45 nt), and where there is no possibility of translation of the RNA transcript. We suggest that the repeated helical opening due to transcriptional initiation is a significant contributor to the induction of high levels of supercoiling. Copyright 1999 Academic Press.