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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.
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PMID:HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae. 158 59

In this report we study the effects of internal deletions of the yeast transcriptional activator HAP1 (CYP1) on activity at two dissimilar DNA binding sites, upstream activation sequence 1 (UAS1) of CYC1 (iso-1-cytochrome c) and CYC7 (iso-2-cytochrome c). These deletions remove up to 1061 amino acids of the 1483-residue protein and bring the carboxyl-terminal acidic activation domain closer to the amino-terminal DNA-binding domain. Surprisingly, the deletions have opposite effects at the two sites; activity at UAS1 increases with deletion size, while activity at CYC7 decreases. The mutant with the largest deletion, mini-HAP1, has no measurable activity at CYC7 but binds normally to the site in vitro. In contrast, a protein with the DNA-binding domain of HAP1 fused to the acidic activation domain of GAL4 is active at both UAS1 and CYC7. These findings are discussed in the context of two models that suggest how the DNA sequence can alter the activity of the bound HAP1. In a separate experiment, we generate a mutation in the DNA-binding domain of HAP1 that requires the addition of zinc for binding to either UAS1 or CYC7 in vitro. This finding shows that a zinc finger anchors DNA binding to both types of HAP1 sites.
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PMID:Internal deletions in the yeast transcriptional activator HAP1 have opposite effects at two sequence elements. 216 46

Heme is a prosthetic group for numerous enzymes, cytochromes and globins, and it binds tightly, sometimes covalently, to these proteins. Interestingly, heme also potentiates binding of the yeast transcriptional activator HAP1 to DNA and inhibits mitochondrial import of the mammalian delta-aminolevulinate synthase (ALAS) and the catalytic activity of the reticulocyte kinase, HRI. All three of these proteins contain a short sequence, the heme regulatory motif (HRM), that occurs six times adjacent to the HAP1 DNA binding domain, twice in the leader targeting sequence of ALAS and twice near the catalytic domain of the HRI kinase. Here we show that a 10 amino acid peptide containing the HRM consensus binds to heme in the micromolar range, and shifts the heme absorption spectrum to a longer wavelength, a direction opposite to the change caused by cytochromes or globins. Further, we show that a single HRM regulates the acidic activation domains of HAP1 and GAL4 independently of regulation of DNA binding of the transcription factors. These findings thus establish a novel heme binding sequence which is structurally distinct from sequences in globins or cytochromes and which has a regulatory function.
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PMID:Heme binds to a short sequence that serves a regulatory function in diverse proteins. 783 42

Various fragments of the N-terminal, DNA-binding domain of the yeast Saccharomyces cerevisiae transcriptional activator CYP1(HAP1) have been cloned and expressed in Escherichia coli. The corresponding polypeptides have been analysed biochemically and we have undertaken a more extensive physical study of a fragment consisting of amino acids 49-126 [CYP1(49-126)]. We show that this CYP1(49-126) peptide requires zinc or cadmium in the growth medium in order to maintain a stable structure. A method to purify CYP1(49-126) is presented. We demonstrate that the purified CYP1(49-126) fragment contains two zinc ions/fragment or two cadmium ions/fragment, which are necessary for DNA binding. 113Cd one-dimensional NMR data suggest that CYP1(HAP1) has a tetrahedral coordination, and that it forms a zinc-cluster complex like GAL4.
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PMID:The DNA-binding domain of the yeast Saccharomyces cerevisiae CYP1(HAP1) transcription factor possesses two zinc ions which are complexed in a zinc cluster. 795 73

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to GAL4, PPR1, and related factors. By selecting random HAP1-binding sites, we found that HAP1, like GAL4, binds to two CGG triplets. Unlike GAL4, the CGGs in the HAP1 consensus are in a direct and not inverted orientation. Sites with inverted CGGs were not recovered, and mutations converting the direct repeat of CGGs to an inverted repeat greatly reduce HAP1-binding affinity. Also, the 6-bp spacer between the CGGs contains a consensus TA that is positioned asymmetrically. Dimethylsulfate protection patterns on six of these sites show protections and enhancements that also lie in a directly repeated orientation, suggesting that the two HAP1 DNA recognition domains of a HAP1 homodimer are oriented in a directly repeated configuration on the DNA. Moreover, substitution of the HAP1 dimerization domain with that of PPR1, which forms coiled-coils and dimerizes symmetrically, did not diminish the ability of the protein to bind selectively to a direct repeat. This result suggests that one DNA-binding domain of the HAP1 homodimer must be able to swivel 180 degrees relative to the dimerization domain to make specific contacts with the second CGG triplet. Our results present a novel example of domain swiveling in one of the two identical subunits of a homodimer to accommodate specific DNA contacts to both CGG triplets of a direct repeat.
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PMID:The yeast activator HAP1--a GAL4 family member--binds DNA in a directly repeated orientation. 795 82

The activity of the yeast transcriptional activator HAP1 is controlled by heme and the heme effect is mediated through the heme domain of HAP1. In this report, we show that HAP1 activity is significantly reduced in strains deleted of TUP1 or SSN6, and addition of a heme analog does not allow HAP1 to regain its full activity. Deletion of the heme domain alleviates the requirement for TUP1/SSN6. The results suggest that TUP1/SSN6 have a positive effect on the activity of HAP1 and this effect is mediated through the heme domain. Although TUP1/SSN6 generally repress transcription of many genes, our data indicate they may have positive effect on the expression of certain genes.
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PMID:Evidence that TUP1/SSN6 has a positive effect on the activity of the yeast activator HAP1. 800 36

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to the zinc finger of GAL4 and an adjacent regulatory domain that blocks DNA binding in the absence of the inducer heme. We show that short HAP1 fragments containing the zinc finger are unable to bind to DNA but can be rescued by antibody to the HAP1 zinc finger. These fragments are missing a coiled-coil sequence similar to that within the dimerization domain of GAL4 and dimerization domains of myosin heavy chain. We surmise that the antibody promotes DNA binding by bringing together two monomers. Interestingly, the antibody will also promote DNA binding of a larger HAP1 fragment containing the DNA-binding and the heme-regulatory domains. This suggests that the regulatory domain acts by preventing dimerization of HAP1 in the absence of heme. Consistent with this view is an in vivo assay that also reveals that heme promotes HAP1 dimerization in yeast cells.
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PMID:Antibody-promoted dimerization bypasses the regulation of DNA binding by the heme domain of the yeast transcriptional activator HAP1. 846 99

The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a transcriptional activator that binds as a homodimer to target DNA sequences. These targets include the upstream activating sequences of the CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though both sites have the same affinity for HAP1, activation differs at these two sites, even when the sequences are placed in an identical promoter context. In addition, mutants of HAP1 that can bind to both sites but are specifically transcriptionally inactive at CYC7 have been previously isolated. In order to identify nucleotides that are responsible for this differential activity, we have performed random and site-directed mutagenesis of these target sites and assayed their binding to HAP1 in vitro and their activity in vivo in reporter plasmids. Our results show that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for activation of the CYC7gene is restored by specific mutations in the CYC7 binding site. Conversely, other mutations of the target sites prevent activation by HAP1, without interfering with DNA binding. The results suggest that the sequence of the target sites influences the conformation and, hence, the activity of DNA-bound HAP1.
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PMID:Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding. 862 77

CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues. Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA. These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases. Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat. Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix. We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1. Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study. This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved. This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix.
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PMID:1H, 15N resonance assignment and three-dimensional structure of CYP1 (HAP1) DNA-binding domain. 868 83

The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.
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PMID:NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein. 922 3


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