Transient and dynamic DNA supercoiling potently stimulates the leu-500 promoter in Escherichia coli

Xiaoduo Zhi,Samantha Dages,Kelley Dages,Yingting Liu,Zi-Chun Hua,John Makemson,Fenfei Leng

doi:10.1074/jbc.m117.794628

Abstract

The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the −10 region of the leucine operon promoter, which causes leucine auxotrophy. This promoter can be activated by (−) DNA supercoiling in Escherichia coli topA strains. However, whether this activation arises from global, permanent, or transient, dynamic supercoiling is still not fully understood. In this article, using a newly established in vivo system carrying a pair of divergently coupled promoters, i.e. an IPTG-inducible promoter and Pleu-500 that control the expression of lacZ and luc (the firefly luciferase gene), respectively, we demonstrate that transient, dynamic (−) DNA supercoiling provided by divergent transcription in both wild-type and topA strains can potently activate Pleu-500. We found that this activation depended on the promoter strength and the length of RNA transcripts, which are functional characteristics of transcription-coupled DNA supercoiling (TCDS) precisely predicted by the twin-supercoiled domain model of transcription in which a (+) supercoiled domain is produced ahead of the RNA polymerase and a (−) supercoiled domain behind it. We also demonstrate that TCDS can be generated on topologically open DNA molecules, i.e. linear DNA molecules, in Escherichia coli, suggesting that topological boundaries or barriers are not required for the production of TCDS in vivo. This work demonstrates that transient, dynamic TCDS by RNA polymerases is a major chromosome remodeling force in E. coli and greatly influences the nearby, coupled promoters/transcription.

Highlights

The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the ؊10 region of the leucine operon promoter, which causes leucine auxotrophy
In this article, using a newly established in vivo system carrying a pair of divergently coupled promoters, i.e. an IPTG-inducible promoter and Pleu-500 that control the expression of lacZ and luc, respectively, we demonstrate that transient, dynamic (؊) DNA supercoiling provided by divergent transcription in both wild-type and topA strains can potently activate Pleu-500
We demonstrate that transcription-coupled DNA supercoiling (TCDS) can be generated on topologically open DNA molecules, i.e. linear DNA molecules, in Escherichia coli, suggesting that topological boundaries or barriers are not required for the production of TCDS in vivo

Summary

Edited by Chris Whitfield

The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the ؊10 region of the leucine operon promoter, which causes leucine auxotrophy. In this article, utilizing different DNA molecules, i.e. circular plasmids, linear plasmids, and the E. coli chromosome, we demonstrate that transient and dynamic (Ϫ) DNA supercoiling provided by transcription from an IPTG-inducible promoter is able to potently activate the divergently coupled supercoilingsensitive promoter Pleu-500 in E. coli cells. Our results showed that TCDS could be generated on topologically open DNA molecules, such as linear DNA templates, to activate Pleu-500 promoter in E. coli These results suggest that topological boundaries or barriers are not required for the production of supercoiled domains around the RNA polymerase in vivo. We found that activation of Pleu-500 by TCDS is dependent on the promoter strength and the length of RNA transcripts, functional properties of TCDS precisely predicted by the twin-domain mechanism [23]

Results

Discussion

Experimental procedures

Plasmid DNA templates

Bacterial strains

Luciferase assay