DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli

Caryn E Outten,F Wayne Outten,Thomas V O'Halloran

doi:10.1074/jbc.274.53.37517

Abstract

MerR-like DNA distortion mechanisms have been proposed for a variety of stress-responsive transcription factors. The Escherichia coli ZntR protein, a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. To determine whether the MerR DNA distortion mechanism is conserved among MerR family members, we have purified ZntR to homogeneity and shown that it is a zinc receptor that is necessary and sufficient to stimulate Zn-responsive transcription at the zntA promoter. Biochemical, DNA footprinting, and in vitro transcription assays indicate that apo-ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to apo-MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. We propose that this zinc-sensing homologue of MerR restructures the target promoter in a manner similar to that of other stress-responsive transcription factors. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal but as yet unknown concentration.

Highlights

Zinc is an essential element that must be maintained at certain levels within all cells
A construct containing a 2-bp1 deletion in the spacer region of the zntA promoter displayed constitutive activity, indicating that the wild-type 20-bp spacing between the Ϫ35 and Ϫ10 sites plays a role in regulation
Promoters regulated by members of the MerR family typically have spacer elements longer than the consensus length of 17 bp which makes them poor substrates for RNA polymerase (RNAP) [31]

Summary

Introduction

Zinc is an essential element that must be maintained at certain levels within all cells. These results led to a model in which apo-MerR bends the DNA toward itself, producing two kinks in the DNA structure that appear as hypersensitive bands within the protected region in DNase I footprinting experiments (see Fig. 1A). Protein/DNA footprinting and in vivo transcriptional assays reveal that, like MerR, ZntR functions primarily as a transcriptional activator, but it only weakly, if at all, represses expression of the zntA gene.

Results

Conclusion