Department of Chemistry & Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA.
Gene. 2013 Feb 10;514(2):82-90. doi: 10.1016/j.gene.2012.11.011. Epub 2012 Nov 29.
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.
RNA 聚合酶的转录可以在体外和体内诱导超负超螺旋 DNA 的形成。这种现象已经被一个“双超螺旋域”转录模型很好地解释了，在这个模型中，一个正超螺旋域在前头，RNA 聚合酶后面是一个负超螺旋域。在大肠杆菌 topA 菌株中，DNA 拓扑异构酶可以选择性地将正超螺旋域转化为负超螺旋，从而产生超负超螺旋 DNA。在本文中，为了研究启动子强度是否影响转录偶联的 DNA 超螺旋化（TCDS），我们开发了一种双质粒系统，其中线性、非超螺旋的质粒用于组成型表达 lac 阻遏物，而圆形质粒用于测量大肠杆菌细胞中的 TCDS。使用这个双质粒系统，我们发现 topA 菌株中的 TCDS 依赖于启动子强度。我们还证明了质粒 DNA 的转录偶联超负超螺旋化不需要强启动子的膜插入蛋白的表达；然而，它可能需要共转录合成多肽。此外，我们发现对于弱启动子，膜插入 tet 基因的表达不足以产生超负超螺旋 DNA。我们的结果可以用“双超螺旋域”转录模型来解释，其中大肠杆菌 RNA 聚合酶所受的摩擦力在超负超螺旋 DNA 的产生中起着关键作用。
