IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins

Abstract

IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S](1+) cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.

Figure 1

Similarity of IscR to MarA. Amino acid sequence alignment of IscR and MarA generated with align showed 20% identical residues (:) and 47% similar residues (.). Solid lines denote the two helix-turn-helix DNA-binding domains of MarA; the three cysteine residues (C92, C98, C104) of IscR that may coordinate the [2Fe-2S] cluster are underlined.

Figure 2

The iscR promoter region. (A) The DNA sequence of the iscR promoter region. The transcription start site identified by 5′ mapping is indicated by an arrow. Matches to an extended −10 element and a consensus −35 hexamer of the Eσ⁷⁰-binding site with a 17-bp spacer region are marked by asterisks. The position of three overlapping marbox-like sequences (sequences that bind MarA) are denoted by the solid lines; −63 to −82 nucleotides contain an exact match to the marbox consensus sequence, whereas the sequences from −38 to −57 nucleotides and −51 to −70 nucleotides match only 9 nucleotides of the 14 conserved marbox nucleotides. (B) The location of the DNA segments used to generate lacZ fusions in this study are shown and consist of the following regions: iscR′ (−476 to +74 nucleotides relative to the iscR start codon), P_iscRiscS′ (−476 to +9 nucleotides relative to the iscR start codon and −226 to +419 nucleotides relative to the iscS start codon), P_iscRiscSUA (−476 +9 nucleotides relative to the iscR start codon and −226 nucleotides relative to the iscS start codon to the end of the iscA ORF). The iscR sequences that were removed to generate the in-frame iscR deletions in the lacZ fusions or in the chromosome (the line flanked by the hash marks) are indicated by parentheses.

Figure 3

IscR negatively regulates expression of iscRSUA. β-Galactosidase activity was measured from strains containing lacZ fusions to the DNA regions shown in Fig. 2. IscR⁺ strains (open bars) contain wild-type iscR, and IscR⁻ strains (filled bars) contain the ΔiscR∷ Kn^R allele; piscR is plasmid pPK5960. The data represent the average activity of three independently isolated strains.

Figure 4

Primer extension assays of iscR mRNA. An autoradiograph of primer-extended RNA products from either MG1655 (iscR⁺) or PK4854 (ΔiscR), by using an iscR-specific primer. Sequencing ladders carried out with the same primer are included as a reference.

Figure 5

EPR spectrum of IscR. EPR spectrum (first derivative) of IscR as obtained on purification under anaerobic conditions. The protein concentration was 1.75 mM, S²⁻1.11 mM and Fe 1.16 mM. The spectra were collected at a frequency of 9.2254 GHz, microwave power of 10 μW, modulation amplitude of 0.4 mT (4 G), modulation frequency of 100 kHz, scan time of 4 min, and temperature of 20 K. The g values are 1.99, 1.93, and 1.88.

Figure 6

IscR represses transcription from the iscR promoter. A phosphorimage of the products from in vitro transcription reactions containing the iscR promoter plasmid, pPK6511 (2 nM) and Eσ⁷⁰ (50 nM) (lanes 1 and 2). The reaction in lane 2 also contained IscR (656 nM protein; 200 nM Fe-S cluster). The arrows indicate the transcripts corresponding to iscR and RNA-1.

Figure 7

IscR activity is decreased in strains lacking Fe-S cluster assembly proteins. β-Galactosidase activity was measured from strains containing a lacZ fusion to the iscR promoter (Fig. 2) and derivatives containing hscA2-cat or ΔiscS∷ Kn^R. The data represent the average activity of three independently isolated strains.