Manipulation of the ‘Zinc cluster’ region of transcriptional activator<i>LEU3</i>be site-directed mutagenesis

Bai, Yuntian Brian; Kohlhaw, Gunter B.

doi:10.1093/nar/19.21.5991

Cited by 28 publications

(28 citation statements)

References 30 publications

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“…As 6. The most prominent feature is the cysteine-rich DNAbinding region, located between positions 37 and 67 (2,32). This region is fully functional, since Leu3p(17-147) binds to UASLEU with an affinity constant that is similar to that of wild-type Leu3p (23a).…”

Section: Resultsmentioning

confidence: 99%

“…The DNA-binding domain is located near the N terminus where residues 37 to 67 conform to the Zn(II)2Cys6 binuclear cluster found in a number of lower eukaryotic DNA-binding proteins, including the well-studied Gal4 protein (Gal4p) (2).…”

mentioning

confidence: 99%

“…Given the fact that the designated DNA contact region of Gal4p, Leu3p, and other lower eukaryotic activators is highly conserved, it is to be expected that their interaction with DNA will follow similar principles. At the same time, there is enough variability both within the zinc-cysteine cluster (especially in its central and C-terminal portions [2]) and in the flanking regions to account for the specificity of the interaction, which may also be aided by helper proteins such as the recently discovered Egdl of S. cerevisiae (22).…”

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confidence: 99%

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Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

Sze¹,

Remboutsika²,

Kohlhaw³

1993

Mol. Cell. Biol.

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The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 (26). Third, in the absence of a-IPM, Leu3p acts as a repressor of transcription (5,26). Leu3p thus appears to be a molecular on-off switch, where "off" means not just the absence of activation, but actual suppression of transcription below basal levels.Three major functions of the 886-residue-long Leu3p (DNA binding, transcriptional activation, and modulation by a-IPM) have been identified, and preliminary assignments for the location of regions responsible for these functions along the primary structure of the protein have been made. The DNA-binding domain is located near the N terminus where residues 37 to 67 conform to the Zn(II)2Cys6 binuclear cluster found in a number of lower eukaryotic DNA-binding proteins, including the well-studied Gal4 protein (Gal4p) (2).

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

Sze¹,

Remboutsika²,

Kohlhaw³

1993

Mol. Cell. Biol.

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“…They include S. cerevisiae Ume6p, as well as C. albicans Czf1p (283). Several mutagenic studies demonstrate the importance of the six cysteine residues in DNA binding and protein function (12,50,79,111,204,205,248,260,293). Other residues found within the metal-binding motif are equally important.…”

Section: Structural and Functional Domainsmentioning

confidence: 99%

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

MacPherson¹,

Larochelle²,

Turcotte

2006

Microbiol Mol Biol Rev

510

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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“…However, mapping of the functional domains along the primary structure of Leu3p has yielded function relationships. Thus, the DNA-binding function of Leu3p is located near the N terminus, where amino acid residues 37 to 67 resemble a motif found in at least a dozen fungal transactivators (3), including the well-studied Gal4 protein. Nuclear magnetic resonance and X-ray crystallographic analyses of the Gal4 protein identified the motif as a Zn(II)2-Cys6 binuclear cluster (9,10,14).…”

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confidence: 99%

Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach.

Remboutsika¹,

Kohlhaw²

1994

Mol. Cell. Biol.

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The Leu3 protein (Leu3p) of Saccharomyces cerevisiae is a pleiotropic transregulator that can function both as an activator and as a repressor of transcription. It binds to upstream promoter elements (UASLEU) with the consensus sequence 5'-GCCGGNNCCGGC-3'. The DNA-binding motif of Leu3p belongs to the family of Zn(II)2-Cys6 clusters. The motif is located between amino acid residues 37 and 67 of the 886-residue protein. In this study, we used a recombinant peptide consisting of residues 17 to 147 to explore the interaction between Leu3p and its cognate DNA. We found that the Leu3p(17-147) peptide is a monomer in the absence of UASLEU but assumes a dimeric structure when the DNA is present. Results of protein-DNA cross-linking and methylation and ethylation interference footprinting experiments show that the Leu3p(17-147) dimer interacts symmetrically with two contact triplets separated by 6 bp and suggest that the peptide approaches its target DNA in such a way that each subunit is positioned closer to one DNA strand than to the other. The binding of Leu3p is strongly affected by the spacing between the contact triplets of the UASLEU and by the type of triplet. Binding occurs when the triplets are 6 bp apart (normal spacing) but fails to occur when the triplets are 0, 5, or 8 bp apart. Weak binding occurs when the triplets are 7 bp apart. Binding does not occur when the UASLEU triplets (GCC....GGC) are replaced with triplets found in the UAS elements for Gal4p, Put3p, and Ppr1p (CGG....CCG). The apparent Kd for the normal Leu3p(17-147)-UASLEU complex is about 3 nM. A mutant form of Leu3p(17-147) in which the histidine at position 50 has been replaced with cysteine binds UASLEU with significantly greater affinity (apparent Kd of about 0.7 nM), even though the interaction between the mutant peptide and target DNA appears to be unchanged. Interestingly, repression of basal-level transcription, which is a hallmark property of the wild-type Leu3p(17-147) peptide, is largely lost with the mutant peptide, indicating that there is no direct correlation between strength of binding and repression.

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Manipulation of the ‘Zinc cluster’ region of transcriptional activatorLEU3be site-directed mutagenesis

Cited by 28 publications

References 30 publications

Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach.

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