Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains

作者：Zhi Xiaoduo; Leng Fenfei^*

来源：Gene, 2013, 514(2): 82-90.

DOI：10.1016/j.gene.2012.11.011

• Transcription-coupled DNA supercoiling
• Promoter strength
• E. coil topA strains
• Twin-supercoiled-domain model of transcription
• Hypernegatively supercoiled DNA

Transcription by RNA polymerase can induce the formation of hypemegatively supercoiled DNA in vitro and in vivo. This phenomenon has been nicely explained by a "twin-supercoiled-domain" model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In Escherichia coli topA strains, DNA gyrase selectively converts the positively supercoiled domain into negative supercoils to produce hypemegatively supercoiled DNA. In this article, in order to examine whether promoter strength affects transcription-coupled DNA supercoiling (TCDS), we developed a two-plasmid system in which a linear, non-supercoiled plasmid was used to express lac repressor constitutively while a circular plasmid was used to gage TCDS in E. coil cells. Using this two-plasmid system, we found that TCDS in topA strains is dependent on promoter strength. We also demonstrated that transcription-coupled hypernegative supercoiling of plasmid DNA did not need the expression of a membrane-insertion protein for strong promoters; however, it might require co-transcriptional synthesis of a polypeptide. Furthermore, we found that for weak promoters the expression of a membrane-insertion tet gene was not sufficient for the production of hypemegatively supercoiled DNA. Our results can be explained by the "twin-supercoiled-domain" model of transcription where the friction force applied to E. coil RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA.

• 出版日期2013-2-10