Visualizing the activity of Escherichia coli divergent promoters and probing their dependence on superhelical density using dual-colour fluorescent reporter vector.

Irina S Masulis,Olga N Ozoline,Zaira Sh Babaeva,Sergey V Chernyshov

doi:10.1038/srep11449

Abstract

Mosaic pattern of transcription in alternating directions is a common feature of prokaryotic and eukaryotic genomes which rationality and origin remain enigmatic. In Escherichia coli approximately 25% of genes comprise pairs of topologically linked divergently transcribed units. Given that transcriptional complex formation at each promoter in the pair induces topological changes and is itself sensitive to DNA structural perturbations, study of the functional anatomy in such areas requires special approaches. Here we suggested the dual-colour promoter probe vector which may become an ideal tool for divergent transcription profiling. The vector was used to characterize the specific genomic region nearby appY with multiple bidirectional promoters predicted in silico. Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes. RNA product transcribed in antisense direction is suggested as a novel RNA. Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

Highlights

It is already well established that global and local variations in superhelical density can affect the rate and efficiency of gene expression in bacteria[1,2]
In order to address how changes in DNA superhelical density may affect divergent transcription, we treated cells with low concentrations of gyrase inhibitor nalidixic acid and monitored activities of two overlapping promoters for fepA and fes genes and promoters of appY-associated “promoter island”. We found that both promoter regions responded to the decrease in negative supercoiling and in both cases changes in the relative activity of divergent promoters were registered, but the scale of effect was greater in the region with multiple promoter-like signals, which assumes its higher topological flexibility
Given that “promoter islands” are more bent and overtwisted than other genomic regions tested so far[12], while the dynamic behavior of DNA is strongly dependent on the equilibrium sequence-dependent trajectory[18], promoter-rich regions may be considered as a natural sensors of global structural perturbations in genomic DNA

Summary

Introduction

It is already well established that global and local variations in superhelical density can affect the rate and efficiency of gene expression in bacteria[1,2]. In order to address how changes in DNA superhelical density may affect divergent transcription, we treated cells with low concentrations of gyrase inhibitor nalidixic acid and monitored activities of two overlapping promoters for fepA and fes genes and promoters of appY-associated “promoter island”. We found that both promoter regions responded to the decrease in negative supercoiling and in both cases changes in the relative activity of divergent promoters were registered, but the scale of effect was greater in the region with multiple promoter-like signals, which assumes its higher topological flexibility. Given that “promoter islands” are more bent and overtwisted than other genomic regions tested so far[12], while the dynamic behavior of DNA is strongly dependent on the equilibrium sequence-dependent trajectory[18], promoter-rich regions may be considered as a natural sensors of global structural perturbations in genomic DNA

Methods

Results

Conclusion