Activating the Prokaryotic leu-500 Promoter by Transient, Dynamic DNA Supercoiling in Escherichia Coli

Abstract

DNA supercoiling is a fundamental property of chromosomal DNA in living cells and greatly affects the efficiency of many essential DNA transactions. For instance, the prokaryotic leu-500 promoter, an inactive promoter in bacteria, and can be activated by supercoiling in the topA strains. Although DNA supercoiling around the promoter region was considered playing an essential role in the activation of the leu-500 promoter, whether the global or the localized supercoiling carries out the activation is not clear. In this study, we developed two in vivo systems to study how transient, dynamic DNA supercoiling (TDDS) activates the leu-500 promoter. The first system consists of E. coli topA strain VS111(DE3) and a linear plasmid. The topA strains provided an IPTG-inducible T7 RNA polymerase for TDDS and also does not have a functional topoisomerase I to remove the (-) supercoiled domain. In this case, the (-) supercoiled domain should exist for longer time. The linear plasmid cannot be globally supercoiled, which provided an excellent template to study TDDS in vivo. We also cloned a luc gene (to express firefly luciferase) under the control of the leu-500 promoter and a lacZ gene under the control of the T7 promoter. Using this unique system, we discovered that TDDS was able to significantly activate the leu-500 promoter. In the second in vivo system, we studied how TDDS activated the lue-500 promoter on the chromosome. Utilizing a transposon Tn7-based method, the divergently coupled Pleu-500 and PT7A1/O4 promoters were inserted to the attTn7 site of the chromosome of MG1655 and VS111. Using these two E. coli strains, we found that TDDS was able to activate Pleu-500 ∼13- and 7-fold in VS111 and MG1655, respectively. Our studies suggest that TDDS has important biological functions in vivo.