Hirota Y, Ohyama T
Division of Molecular Biology, Meiji Institute of Health Science, Odawara, Japan.
J Mol Biol. 1995 Dec 8;254(4):566-78. doi: 10.1006/jmbi.1995.0639.
This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the -35 consensus sequence of Escherichia coli promoters. A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately upstream of the beta-lactamase promoter in plasmid pUC19. All the mutants showed reduced promoter activities in E. coli cells. However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment. Leftward writhe greatly repressed transcription. A plane curve showed an intermediate effect. This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter. The positive effect of rightward writhe was also observed in vivo for the promoter of the tetracycline resistance gene of pBR322. The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity.
本研究探讨了位于大肠杆菌启动子 -35 共有序列紧邻上游区域的超螺旋扭曲对转录的影响。设计、合成了一组双链寡核苷酸，每个都具有独特的 DNA 构型，并将其替换到质粒 pUC19 中β-内酰胺酶启动子紧邻上游的天然存在的右手超螺旋曲率区域。所有突变体在大肠杆菌细胞中均表现出启动子活性降低。然而，与直的 DNA 片段产生的效果相比，向右的超螺旋扭曲明显促进了转录。向左的扭曲则极大地抑制了转录。平面曲线显示出中间效应。这种现象不仅是由于该片段驱动开放复合物形成能力的差异，还由于 RNA 聚合酶与启动子结合亲和力的差异。在体内对 pBR322 的四环素抗性基因启动子也观察到了向右扭曲的积极作用。DNA 曲率的超螺旋扭曲的方向和程度似乎决定了其对启动子活性的影响。
