The OmpR protein of Escherichia coli binds to sites in the ompF promoter region in a hierarchical manner determined by its degree of phosphorylation.

A Rampersaud,S.L Harlocker,M Inouye

doi:10.1016/s0021-9258(18)99912-6

A Rampersaud, S.L Harlocker + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)99912-6

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Abstract

In Escherichia coli the ompF gene encodes a major outer membrane porin protein that is differentially regulated by the OmpR protein. OmpR acts as a positive as well as a negative regulator of ompF expression by binding to DNA sequences in the ompF promoter region. The DNA binding activity of OmpR is itself regulated by phosphorylation through the kinase protein EnvZ. Phosphorylation is believed to change the function of OmpR from an activator to a repressor molecule. By using purified OmpR and various regions of the ompF promoter we show that phosphorylation causes binding of OmpR to a DNA region between the -40 to -100 region of the ompF promoter previously shown to be important for ompF expression. As the amount of OmpR-phosphate increases, a binding site located at a further upstream -360 to -380 region was occupied. This latter site has been reported to be important for ompF repression. Further experiments indicate that the -70 to -100 region is a high affinity site, while the -45 to -60 and -360 to -380 regions are low affinity sites. We also provide evidence that OmpR binding at the -360 to -380 region requires previous binding at downstream sequences, which is indicative of long range interactions between OmpR molecules. We interpret our results in terms of a model for ompF regulation involving hierarchical binding by phosphorylated OmpR and potential DNA looping.

Highlights

In Eecherichia coli the ompF gene encodes a major and increase the relative amounts of OmpR-phosphate
Fur- The -70 to -100 region is essential for ompF activation and ther experiments indicate that th-e70 to -100 regionis contains three tandemly arrange1d0-bp' elements, which share sequence similarities with each regions are low affinity sites.We provide evidence other(8,211
The -70 to -100 region, is essential for ompF activation, while the -42 to -52 and -360 to -380regions represent binding sites from which OmpRfacilitates negative regulation

Summary

None of these partially purified forms of OmpR required in

The higher molecular weight of the a-complex is dueto fur- vitro phosphorylation by EnvZ for their DNA binding activities, ther OmpR interactions at the -40 to -60 region (see below). suggesting thatthey were phosphorylated before purification. At a n 8-fold molar excess, this region caused a the relativeDNA binding activities observed by in vitro minor change in the ratioosf a- to b-complexes but otherwise phosphorylation of OmpR are comparable with those obtained did not significantly compete either thea- or b-complex. These from in vivo phosphorylated forms of the protein. -100 region before it can be bound This aspectis developed in -60 region [9, 21].

Hierarchical Binding of OmpR tothe ompF Promoter

DNase Z Footprinting of the Upstream Region of the ompF

DISCUSSION

Fa Fb FC