Supercoiling Effects on Short-Range DNA Looping in E. coli

Lauren S Mogil,Nicole A Becker,L James Maher

doi:10.1371/journal.pone.0165306

Abstract

DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6–8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (ΔtopA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70–85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria.

Highlights

DNA looping is a fundamental mechanism for the control of gene expression in prokaryotes [1, 2] and eukaryotes [3, 4]
The E. coli lac operon provides a classic model for understanding control of gene expression by DNA looping [5,6,7,8,9,10] and for measuring DNA flexibility in vivo [11,12,13,14]
We study control of the endogenous wild type lac operon and apply an adaptation of the experimental system previously developed by Becker et al to study DNA flexibility in living E. coli [11, 40, 41]

Summary

Introduction

DNA looping is a fundamental mechanism for the control of gene expression in prokaryotes [1, 2] and eukaryotes [3, 4]. Three bacterial strains [38, 39] are used in this study to test if the extent of negative supercoiling affects DNA looping. Strain RS2 carries partially defective topoisomerase mutant ΔtopA10 resulting in increased negative DNA supercoiling relative to wild type cells.

Results

Conclusion