Effect of FIS on Transcription-Coupled DNA Supercoiling in E. coli

Abstract

For decades, it has been recognized that transcribing along the DNA double helix by a RNA polymerase can enhance localized DNA supercoiling. This process has been elegantly explained by a “twin-supercoiled-domain” model of transcription in which positive DNA supercoils are generated ahead of a translocating RNA polymerase and negative supercoils behind it. In this study, we utilized bacterial genetics and biochemical approaches to investigate how FIS protein regulates transcription-coupled DNA supercoiling (TCDS) by the strong rrnB P1, P2 promoters. We first generated several E. coli strains including MG1655(DE3)ΔfisΔlacZ and VS111(DE3)ΔfisΔlacZ using the λ Red recombination system. We then inserted the divergently coupled Pleu-500 and IPTG-inducible rrnB P1, P2 promoters with the luc and lacZ genes to the attTn7 site of the E. coli chromosome. Additionally, the upstream region of the rrnB P1 promoter including the three FIS-binding sites was placed between the Pleu-500 and the rrnB P1, P2 promoters. Our results showed that TCDS initiated from the rrnB P1, P2 promoters greatly activated the divergently coupled Pleu-500 promoter. Intriguingly, we found that FIS protein activated the transcription from the P1 and P2 promoters and potently inhibited the activation of the Pleu-500 promoter by TCDS. Our results are consistent with a model in which FIS protein forms topological barriers upon binding to its recognition sites in the upstream region of the rrnB P1 promoter, blocks TCDS diffusion, and potently inhibits the activation of the Pleu-500 promoter by TCDS. Likewise, TCDS generated from the strong P1 and P2 promoters bounced back and activated the P1 and P2 promoters. We also used atomic force microscopy to examine the shape and volume of these E. coli mutant strains.