SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene<i>smtA</i>: identification of a Zn inhibited DNA-protein complex

Morby, Andrew P.; Turner, Jennifer S.; Huckle, James W.; Robinson, Nigel J.

doi:10.1093/nar/21.4.921

Cited by 164 publications

(111 citation statements)

References 18 publications

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“…These sites are thought to have arisen as a result of convergent evolution due to evolutionary pressures (12), a finding consistent with the ''rule of varied allosteric control'' in which protein families evolve seemingly random allosteric control pathways (13). CzrA and its homolog SmtB in Synechococcus (14) are ␣5 sensors that bind Zn(II) ions in two rotationally symmetric tetrahedral coordination sites formed by pairs of metal ligands derived from the ␣5 helix of each subunit (8). The crystal structure of CzrA and SmtB in the apo and the Zn(II)-bound states have been solved (8).…”

Section: Staphylococcus Aureussupporting

confidence: 56%

Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

Arunkumar

Campanello

Giedroc

2009

Proc. Natl. Acad. Sci. U.S.A.

180

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Staphylococcus aureusCzrA is a zinc-dependent transcriptional repressor from the ubiquitous ArsR family of metal sensor proteins. Zn(II) binds to a pair of intersubunit C-terminal ␣5-sensing sites, some 15 Å distant from the DNA-binding interface, and allosterically inhibits DNA binding. This regulation is characterized by a large allosteric coupling free energy (⌬Gc) of approximately ؉6 kcal mol ؊1 , the molecular origin of which is poorly understood. Here, we report the solution quaternary structure of homodimeric CzrA bound to a palindromic 28-bp czr operator, a structure that provides an opportunity to compare the two allosteric ''end'' states of an ArsR family sensor. Zn ( M etal ion homeostasis is a complex process that involves maintaining a delicate balance between metal uptake/ efflux and other metal storage systems to meet the needs of the cell (1, 2). This process is tightly regulated at the level of transcription by means of specific metal-dependent transcriptional regulators that respond to changes in metal ion concentrations in the host environment (3). In Staphylococcus aureus, the czr operon encodes a CDF antiporter, CzrB, a homolog of Escherichia coli zinc transporter YiiP that confers resistance to Zn(II) and Co(II) (4, 5), and the metal-regulated repressor, CzrA (6, 7). CzrA binds Zn(II) with picomolar affinity and strong negative homotropic cooperativity (8, 9) and is thought to undergo a conformational change that alleviates transcriptional repression of the resistance gene czrB.CzrA belongs to the ubiquitous ArsR (or ArsR/SmtB) family of metalloregulators found in many bacterial genomes that sense a wide variety of metals including biologically essential metals as well as toxic metal pollutants (10, 11). Members of this family appear to adopt a common winged helix-turn-helix homodimeric fold, but have evolved physically and structurally distinct pairs of allosteric metal-sensing sites (2, 12). These sites are thought to have arisen as a result of convergent evolution due to evolutionary pressures (12), a finding consistent with the ''rule of varied allosteric control'' in which protein families evolve seemingly random allosteric control pathways (13). CzrA and its homolog SmtB in Synechococcus (14) are ␣5 sensors that bind Zn(II) ions in two rotationally symmetric tetrahedral coordination sites formed by pairs of metal ligands derived from the ␣5 helix of each subunit (8). The crystal structure of CzrA and SmtB in the apo and the Zn(II)-bound states have been solved (8). Although the structures of CzrA were found to be very similar in these two states, SmtB revealed measurable differences in quaternary structure in which the apo form adopted a comparatively ''flat'' conformation not well suited to interact with canonical B-form DNA (8,15). The structural and thermodynamic underpinnings of metalloregulation for any member of the ubiquitous ArsR family remains poorly understood due to a lack of detailed insight for the DNA operator-bound state (3).We report here the NMR solution structure of ...

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Section: Staphylococcus Aureussupporting

confidence: 56%

Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

Arunkumar

Campanello

Giedroc

2009

Proc. Natl. Acad. Sci. U.S.A.

180

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“…The SmtB protein, which represses metallothionein expression in the cyanobacterium Synechococcus sp. strain PCC7942, is a helix-turn-helix protein whose DNA binding ability is inhibited by zinc binding to the protein (8,22). Zinc-induced expression of metallothionein genes in mammals involves the binding of one or more metal-responsive transcription factors (e.g., MTF-1 or ZRF) to sites (metal-regulated enhancer elements) in the metallothionein gene promoters (21,34).…”

Section: Discussionmentioning

confidence: 99%

Zap1p, a Metalloregulatory Protein Involved in Zinc-Responsive Transcriptional Regulation in Saccharomyces cerevisiae

Zhao

Eide

1997

Molecular and Cellular Biology

251

260

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Zinc ion homeostasis in Saccharomyces cerevisiae is controlled primarily through the transcriptional regulation of zinc uptake systems in response to intracellular zinc levels. A high-affinity uptake system is encoded by the ZRT1 gene, and its expression is induced more than 30-fold in zinc-limited cells. A low-affinity transporter is encoded by the ZRT2 gene, and this system is also regulated by zinc. We used a genetic approach to isolate mutants whose ZRT1 expression is no longer repressed in zinc-replete cells, and a new gene, ZAP1, was identified. ZAP1 encodes a 93-kDa protein with sequence similarity to transcriptional activators; the C-terminal 174 amino acids contains five C 2 H 2 zinc finger domains, and the N terminus (residues 1 to 706) has two potential acidic activation domains. The N-terminal region also contains 12% histidine and cysteine residues. The mutant allele isolated, ZAP1-1 up , is semidominant and caused high-level expression of ZRT1 and ZRT2 in both zinc-limited and zinc-replete cells. This phenotype is the result of a mutation that substitutes a serine for a cysteine residue in the N-terminal region. A zap1 deletion mutant grew well on zinc-replete media but poorly on zinc-limiting media. This mutant had low-level ZRT1 and ZRT2 expression in zinc-limited as well as zinc-replete cells. These data indicate that Zap1p plays a central role in zinc ion homeostasis by regulating transcription of the zinc uptake system genes in response to zinc. Finally, we present evidence that Zap1p regulates transcription of its own promoter in response to zinc through a positive autoregulatory mechanism.Zinc is essential for all organisms. This metal is a catalytic component of over 300 enzymes, including alcohol dehydrogenase, carbonic anhydrase, and carboxypeptidases (33). Zinc also plays a structural role in many proteins. For example, several motifs found in transcriptional regulatory proteins are stabilized by zinc, including the zinc finger, zinc cluster, and RING finger domains (28). Proteins containing these domains are very common; as many as 1% of all human gene products contain the C 2 H 2 zinc finger motif first identified in transcription factor IIIA (TFIIIA) (18).Although it is essential for many different cellular functions, zinc can also be toxic. When the intracellular zinc level rises to some critical level, the metal can interfere with vital processes, perhaps by competing with other metal ions for enzyme active sites, transporter proteins, and other biologically important ligands. The delicate balance of intracellular zinc is accomplished through precise homeostatic regulation mediated by a number of mechanisms. These include binding of the metal by cytoplasmic macromolecules such as metallothioneins (14) and phytochelatins (27), zinc storage in intracellular compartments (3,20,25), and transport of the metal out of the cell (26).The primary control point for zinc ion homeostasis is the regulation of zinc uptake across the plasma membrane. Little is known about the mechanism and regulat...

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“…The other members of this family are ArsR (the regulatory protein for the arsenic resistance system [4,12,15,23]) and SmtB (the regulatory protein for cyanobacterial metallothionein synthesis [6]). Bairoch (1) suggests that a helix-turn-helix motif is a recognizable candidate for the DNA binding region.…”

Section: Discussionmentioning

confidence: 99%

CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

1995

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, and Pb 2؉ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions ؊7 to ؉14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro.The cadmium resistance system of Staphylococcus aureus plasmid pI258 was identified by Novick and Roth (9) and subsequently mapped by deletion analysis (8). The cadA system results in reduced net uptake of Cd 2ϩ because of increased energy-dependent efflux of the toxic cation (22). Cloning and DNA sequence analysis identified two genes called cadC and cadA (10). The cadA gene product is a P-type ATPase, a member of the large family of related cation transport ATPases found in plant and animal cells as well as in microbes (16, While the properties of CadA satisfactorily explain cadmium resistance, the role of cadC and the nature of the trans-acting component of the carefully regulated CadA system (3, 25) remain unclear. The cadC gene is involved in full cadmium resistance; cadC can be provided in trans and need not be present on the same plasmid as cadA (26). The inducible nature of transcription was established, and the initiation site for cad mRNA synthesis was identified by reverse transcriptase primer extension (25) to a position 28 nucleotides upstream of the ATG start codon of the cadC gene (Fig. 1). Operon-length mRNA of 2.6 kb, including both cadC and cadA, was identified by Northern blot DNA-RNA hybridization (25). Canonical Ϫ10 and Ϫ35 Escherichia coli-like RNA polymerase recognition sites (Fig. 1) were identified in the sequence (25). A 7-nucleotide inverted repeat segment, including the first nucleotides transcribed into mRNA, was proposed as the operator site for binding of a then-elusive trans-acting DNA-binding regulatory protein (25).Cation-inducible gene function was studied by measurements of Cd 2ϩ efflux by whole cells (25) and subcellular membranes (21) and through induction of the reporter genes ␤-lactamase (25) and luciferase (3). However, the presumed transacting regulatory protein was not identified (25). Because of the need for cadC in gene regulation (21) and the sequence homologies (1, 26) among the predicted CadC protein product, the ArsR regulatory protein (12,14,15,23) for the arsenic resistance system, and the SmtB regulatory protein for cyanobacterial metallothionein synthesis (6), we undertook a direct test of the hypothesis that CadC is the regulatory protein for cadA operon transcription. By in vitro assays with CadC protein and the appropriate operator/promoter DNA, CadC has been shown to be a DNA-binding, negatively acting regulatory protein. MATERIALS AND METHODSAbbreviations. DTT, dithiothreitol; IPTG, isopropyl-␤-D-thiogalactopyranosid...

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SmtB is a metal-dependent repressor of the cyanobacterial metallothionein genesmtA: identification of a Zn inhibited DNA-protein complex

Cited by 164 publications

References 18 publications

Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

Zap1p, a Metalloregulatory Protein Involved in Zinc-Responsive Transcriptional Regulation in Saccharomyces cerevisiae

CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

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