The Solution Structure of an AlcR-DNA Complex Sheds Light onto the Unique Tight and Monomeric DNA Binding of a Zn2Cys6 Protein

Cahuzac, Bertrand; Cerdan, Rachel; Felenbok, Béatrice; Guittet, Éric

doi:10.1016/s0969-2126(01)00640-2

Cited by 23 publications

(24 citation statements)

References 39 publications

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“…As we have recently shown, the Arg6 residue is not directly involved in nuclear transport but rather is involved in DNA target recognition (38). An individual substitution of either Arg5 or Arg7 which, in contrast, do not contribute to the DNA-binding affinity (3,38), did not result in a drastic accumulation of the GFP fusion in the cytoplasm (results not shown). However, when all three arginines were changed simultaneously, an additive effect was observed, which may reflect a complex network of interactions between the NLS sequence of AlcR and its transport receptors.…”

supporting

confidence: 53%

“…The AlcR N terminus plays an additional role via the involvement of Arg6 in the specificity of binding to palindromic sites and in transcriptional activation (38). The three-dimensional structure of the AlcR DNA-binding domain (amino acids [aa] 1 to 60), solved by nuclear magnetic resonance, has provided evidence that, indeed, contacts between the N-terminal arm of AlcR occur in the minor groove of DNA through this Arg6 residue (3).…”

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confidence: 99%

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Nuclear Import of Zinc Binuclear Cluster Proteins Proceeds through Multiple, Overlapping Transport Pathways

2003

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In Aspergillus nidulans, the high transcriptional level of the ethanol utilization pathway genes (alc) is regulated by the specific activator AlcR. Here we have analyzed the mechanism of the nuclear import of AlcR, as well as that of other proteins belonging to the Zn 2 Cys 6 binuclear cluster family. The nuclear localization signal of AlcR maps within the N-terminal 75 amino acid residues and overlaps with its DNA-binding domain. It consists of five clusters rich in basic residues. Four of them are necessary and sufficient for nuclear targeting. The first two basic regions are crucial for both nuclear localization and recognition of AlcR-specific DNA targets. This nuclear localization signal (NLS) motif is recognized by the nuclear transport machinery of Saccharomyces cerevisiae and requires both Ran/Gsp1p activity and specific transport receptors. AlcR can be imported into nuclei via multiple transport pathways mediated by a distinct set of karyopherins composed of Kap104p, Sxm1p, and Nmd5p transport receptors. The two former karyopherins interact with the NLS of AlcR directly. Other Zn binuclear cluster proteins from S. cerevisiae, such as Gal4p and Pdr3p, also appear to be transported to the nuclei in a nonclassical, importin-␣-independent manner and can share common importin ␤ receptors.Transport of macromolecules between the cytoplasm and the nucleus in eukaryotic cells generally proceeds via soluble transport receptors that bind to specific nuclear localization signals (NLSs) on their substrates and translocate them across the nuclear membrane through specific structures called nuclear pore complexes (NPC) (reviewed in references 18 and 36). Several distinct transport pathways have been identified thus far, most mediated by different members of the importin ␤ receptor family (reviewed in references 7 and 36). Of these, the most extensively characterized is the pathway that involves import of proteins with classical NLS composed of one or two clusters of basic residues. Nuclear import of such proteins is initiated by binding to the importin ␣/␤ heterodimer, where importin ␣ acts as an adapter subunit to bridge NLS cargoes to importin ␤, which transports the whole complex through the nuclear envelope (16). Importin ␣ (also known as karyopherin ␣ in vertebrates or Kap60p/Srp1p in Saccharomyces cerevisiae) directly interacts with the NLS via its armadillo-like repeated motifs (7, 9). In turn, its highly basic, N-terminal importin ␤ binding (IBB) domain is recognized by the importin ␤ receptor (also called karyopherin ␤ in vertebrates or Kap95p/Rsl1p in yeast) (8). Once formed, the resultant heterotrimeric complex is recruited to the NPC through the interaction of the importin ␤ subunit with the NPC components (reviewed in references 12 and 18). Subsequent translocation of the docked complex into the nuclear compartment requires the integrity of the small Ras-related GTP binding protein Ran (the yeast homolog Gsp1p) and several Ran-associated proteins (21, 50). The dissociation of the importin heterodimer foll...

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confidence: 53%

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confidence: 99%

Nuclear Import of Zinc Binuclear Cluster Proteins Proceeds through Multiple, Overlapping Transport Pathways

2003

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“…Our EMSA analysis suggests that Ert1(1-152) binds as a monomer. Although binding of a zinc cluster protein to DNA as monomer has been reported (Cahuzac et al 2001), it is quite possible that Ert1 binds to DNA as a homoor a heterodimer. For example, the DNA binding domain used in our studies may lack a dimerization domain.…”

Section: Discussionmentioning

confidence: 99%

The Switch from Fermentation to Respiration in Saccharomyces cerevisiae Is Regulated by the Ert1 Transcriptional Activator/Repressor

et al. 2014

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In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene. IN the yeast Saccharomyces cerevisiae, glucose is the preferred carbon source and fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, a process requiring a shift to a respiration mode. Other nonfermentable carbon sources, such as lactate, acetate, or glycerol, can also be used by yeast . The shift from fermentative to nonfermentative metabolism results in massive reprogramming of gene expression for the use of ethanol (Derisi et al. 1997;Roberts and Hudson 2006). For example, increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced under these conditions. An important player in the regulation of this process is the Snf1 kinase (Hedbacker and Carlson 2008;Zaman et al. 2008;Zhang et al. 2010;Broach 2012). Snf1 becomes activated under low glucose conditions resulting in the phosphorylation of a number of substrates that include DNA binding proteins such as Mig1, Cat8, Sip4, and Rds2 (for reviews see Schüller 2003;Turcotte et al. 2011).Mig1 and Adr1 belong to the family of zinc finger proteins of the Cys 2 His 2 type. Mig1 is a transcriptional repressor that, following phosphorylation by Snf1, undergoes nucleocytoplasmic shuffling...

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“…A classic example of a monomer is the Aspergillus AlcR protein. NMR spectroscopy clearly shows that one monomeric zinc cluster binds to an element in the alcA promoter, which contains the sequence CGTGCGGATC (28). Monomer or dimer status can sometimes be inferred based on the sequence of its target regulatory element.…”

Section: Mechanisms Of Actionmentioning

confidence: 99%

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

MacPherson¹,

Larochelle²,

Turcotte

2006

Microbiol Mol Biol Rev

502

554

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SUMMARY The trace element zinc is required for proper functioning of a large number of proteins, including various enzymes. However, most zinc-containing proteins are transcription factors capable of binding DNA and are named zinc finger proteins. They form one of the largest families of transcriptional regulators and are categorized into various classes according to zinc-binding motifs. This review focuses on one class of zinc finger proteins called zinc cluster (or binuclear) proteins. Members of this family are exclusively fungal and possess the well-conserved motif CysX2CysX6CysX5-12CysX2CysX6-8Cys. The cysteine residues bind to two zinc atoms, which coordinate folding of the domain involved in DNA recognition. The first- and best-studied zinc cluster protein is Gal4p, a transcriptional activator of genes involved in the catabolism of galactose in the budding yeast Saccharomyces cerevisiae. Since the discovery of Gal4p, many other zinc cluster proteins have been characterized; they function in a wide range of processes, including primary and secondary metabolism and meiosis. Other roles include regulation of genes involved in the stress response as well as pleiotropic drug resistance, as demonstrated in budding yeast and in human fungal pathogens. With the number of characterized zinc cluster proteins growing rapidly, it is becoming more and more apparent that they are important regulators of fungal physiology.

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The Solution Structure of an AlcR-DNA Complex Sheds Light onto the Unique Tight and Monomeric DNA Binding of a Zn2Cys6 Protein

Cited by 23 publications

References 39 publications

Nuclear Import of Zinc Binuclear Cluster Proteins Proceeds through Multiple, Overlapping Transport Pathways

Nuclear Import of Zinc Binuclear Cluster Proteins Proceeds through Multiple, Overlapping Transport Pathways

The Switch from Fermentation to Respiration in Saccharomyces cerevisiae Is Regulated by the Ert1 Transcriptional Activator/Repressor

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

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