title: Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains.
abstract: Transcription by RNA polymerase can induce the formation of hypernegatively 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 hypernegatively 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. coli 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 hypernegatively supercoiled DNA. Our results can be explained by the "twin-supercoiled-domain" model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA.
authors.affiliation: Department of Chemistry & Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA.
authors.forename: Xiaoduo
authors.initials: X
authors.lastname: Zhi
authors.forename: Fenfei
authors.initials: F
authors.lastname: Leng
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D004264:DNA Topoisomerases, Type I