Miscoordination of the Iron-Sulfur Clusters of the Anaerobic Transcription Factor, FNR, Allows Simple Repression but Not Activation

Colin Scott,Jeffrey Green

doi:10.1074/jbc.m106192200

Colin Scott, Jeffrey Green

Open Access

https://doi.org/10.1074/jbc.m106192200

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Abstract

The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.

Highlights

The transition from aerobic to anaerobic respiration in the facultative anaerobe Escherichia coli is, in part, governed by the global transcription factor FNR [1, 2]
Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively
At class I promoters, AR1 of the downstream subunit of the FNR dimer contacts the C-terminal domain of the ␣-subunit of RNA polymerase (RNAP) [10, 11]

Summary

Introduction

The transition from aerobic to anaerobic respiration in the facultative anaerobe Escherichia coli is, in part, governed by the global transcription factor FNR (the regulator of fumarate and nitrate reduction) [1, 2]. Signal Perception and Transduction in FNR for the C20S substitution, allowing in vivo activation of expression from FNR-dependent promoters These observations suggest that FNR-C20S can assemble oxygen-labile [4Fe-4S] clusters in vivo and that the presence of the miscoordinated ironsulfur clusters promotes dimerization and DNA binding but not the conformational changes required for effective contact with RNAP and transcription regulation [16]. It is concluded that the model describing the aerobic-anaerobic FNR switch can be refined such that iron-sulfur cluster assembly by two FNR monomers permits dimerization and enhanced DNA binding but that only correctly liganded iron-sulfur clusters can cause the necessary conformational changes to establish effective contacts with RNAP and subsequent transcription activation

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