NMR solution structure of AlcR (1–60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein

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

doi:10.1006/jmbi.1999.3417

Cited by 8 publications

(8 citation statements)

References 29 publications

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“…This is in contrast to other Zn cluster proteins, which make strong contacts with two or more G residues in both strands of DNA (33). DMS footprinting (20) and nuclear magnetic resonance structural analysis of AlcR (6) revealed that the protein contacts two G residues (underlined) of the CGG triplet (5Ј-CGG-3Ј). Ppr1, a dimeric Zn cluster protein with almost the same sequence of the conserved recognition helix (5Ј-CRLKKIKC-3Ј) as Rgt1 (5Ј-CRKKKIKC-3Ј), directly interacts with the two G residues of the CGG triplet (22).…”

Section: Discussionmentioning

confidence: 74%

“…The binding sites of the different zinc cluster-containing proteins differ in the orientation of the CGG sequences and in the number of nucleotides separating them. For example, in the binding sites of Gal4, Ppr1, and Put3, the pair of CGG sequences is inverted (5Ј-CGG-CCG-3Ј) and separated by 11,6, and 10 bp, respectively (21,22,31). Leu3 binds to an everted pair of CGG sequences (5Ј-CCG-CGG-3Ј) that are separated by 4 bp (15).…”

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confidence: 99%

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Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

Kim

Polish

Johnston

2003

Molecular and Cellular Biology

118

165

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Rgt1 is a glucose-responsive transcription factor that binds to the promoters of several HXT genes encoding glucose transporters in Saccharomyces cerevisiae and regulates their expression in response to glucose. Rgt1 contains a Zn 2 Cys 6 binuclear cluster responsible for DNA binding. Most proteins that contain this sequence motif bind as dimers to regularly spaced pairs of the sequence CGG. However, there are no CGG pairs with regular spacing in promoters of genes regulated by Rgt1, suggesting that Rgt1 binds as a monomer to CGG or to another sequence. We identified the Rgt1 consensus binding site sequence 5-CGGANNA-3, multiple copies of which are present in all HXT promoters regulated by Rgt1. Rgt1 binds in vivo to multiple sites in the HXT3 promoter in a nonadditive, synergistic manner, leading to synergistic repression of HXT3 transcription. We show that glucose inhibits the DNA-binding ability of Rgt1, thereby relieving repression of HXT gene expression. This regulation of Rgt1 DNA-binding activity is caused by its glucose-induced phosphorylation: the hyperphosphorylated Rgt1 present in cells growing on high levels of glucose does not bind DNA in vivo or in vitro; dephosphorylation of this form of Rgt1 in vitro restores its DNA-binding ability. Furthermore, an altered Rgt1 that functions as a constitutive repressor remains hypophosphorylated when glucose is added to cells and binds DNA under these conditions. These results suggest that glucose regulates the DNA-binding ability of Rgt1 by inducing its phosphorylation.

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Section: Discussionmentioning

confidence: 74%

mentioning

confidence: 99%

Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

Kim

Polish

Johnston

2003

Molecular and Cellular Biology

118

165

View full text Add to dashboard Cite

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“…This model, generalizing the binding pattern of Gal4p‐like proteins, is inadequate to account for other existing modes of binding (Strauss et al , 1998; King et al , 1999; Nikolaev et al , 1999; Cerdan et al , 2000; Cahuzac et al , 2001). Here, we describe a protein that, although binding preferentially to CCGG‐N‐CCGG inverted repeats, shows striking plasticity.…”

Section: Discussionmentioning

confidence: 99%

“…AlcR presents a unique mode of binding. It binds as a monomer, and both mutational and nuclear magnetic resonance (NMR) studies show that residues outside the first basic loop are essential for sequence recognition (Nikolaev et al , 1999; Cerdan et al , 2000; Cahuzac et al , 2001). The FacB activator recognizes two sets of very different sequences, both possibly imperfect inverted repeats (Todd et al , 1998), but no details are known about its mode of binding.…”

Section: Introductionmentioning

confidence: 99%

PrnA, a Zn₂Cys₆ activator with a unique DNA recognition mode, requires inducer for in vivo binding

Gómez

Cubero

Cecchetto

et al. 2002

Molecular Microbiology

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Summary The PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG‐N‐CCGG inverted repeats and to CCGG‐6/7N‐CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG‐6N‐CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG‐N‐CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino‐terminal to it. Three high‐affinity sites in two different promoters have been visualized by in vivo methy‐lation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high‐affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiæ protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.

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“…The recent solution of the NMR structure of the AlcR DNA‐binding domain provides new features concerning differences between this protein and other known zinc clusters (Cerdan et al 2000). Moreover, modeling the AlcR–DNA complex by homology with the GAL4–DNA complex suggests that a tryptophan residue could possibly interact with a thymine (or adenine).…”

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confidence: 99%

Differential chemical labeling of the AlcR DNA‐binding domain from Aspergillas nidulans versus its complex with a 16‐mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

et al. 2001

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DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

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NMR solution structure of AlcR (1–60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein

Cited by 8 publications

References 29 publications

Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

PrnA, a Zn₂Cys₆ activator with a unique DNA recognition mode, requires inducer for in vivo binding

Differential chemical labeling of the AlcR DNA‐binding domain from Aspergillas nidulans versus its complex with a 16‐mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

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NMR solution structure of AlcR (1–60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein

Cited by 8 publications

References 29 publications

Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding

Differential chemical labeling of the AlcR DNA‐binding domain from Aspergillas nidulans versus its complex with a 16‐mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

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PrnA, a Zn₂Cys₆ activator with a unique DNA recognition mode, requires inducer for in vivo binding