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

The yeast transcriptional activator GAL4 binds co-operatively to four related 17-base-pair sequences within an upstream activating sequence (UASG) to activate transcription of the GAL1 and GAL10 genes. It belongs to a class of gene regulatory proteins which all contain a highly conserved cysteine-rich region within their DNA-binding domains. This region binds zinc and it has been proposed that the cysteine residues coordinate the zinc, creating a structure analogous to one of the 'zinc fingers' of the transcription factor TFIIIA (ref. 8). Using 1H-113Cd two-dimensional nuclear magnetic resonance spectra of the cadmium form of the domain, we previously showed that the protein contains a Cd2Cys6 cluster where cysteines 11 and 28 act as bridging ligands. A similar study of a fragment of GAL4 has recently been published. We report here the solution structure of the DNA binding domain of GAL4; two helix-turn-strand motifs pack around a Zn2Cys6 cluster in a novel pseudo-symmetrical arrangement. The results show that the GAL4 zinc-binding domain differs significantly from both the TFIIIA-type zinc finger and the steroid hormone receptor DNA-binding domains.
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PMID:Structure of the DNA-binding domain of zinc GAL4. 155 14

We describe a genetic system for monitoring the activity of a specific proteolytic enzyme by taking advantage of the properties of the yeast transcriptional activator GAL4. The GAL4 protein contains two separable and functionally essential domains: the amino-terminal DNA binding domain and the carboxyl-terminal transcriptional activating domain. We constructed two hybrid proteins by inserting between the DNA binding domain and the activation domain of GAL4 either (i) a self-cleaving protease (3C protease of a picornavirus, coxsackievirus B3) or (ii) a mutant form of the protease that is unable to cleave. We show that, although the hybrid protein containing the mutant protease activates transcription of GAL1-lacZ reporter gene, the hybrid protein bearing the wild-type protease is proteolytically cleaved and fails to activate transcription. Our approach to monitor the proteolytic activity could be used to develop simple genetic systems to study other proteases.
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PMID:A genetic system for studying the activity of a proteolytic enzyme. 157 Mar 42

We have previously shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can activate a synthetic promoter containing consensus-binding sites for the cellular transcription factor Sp1. In this report, we show that a GAL-Tat fusion protein targeted via GAL4 DNA-binding sites can also trans activate an HIV-1 LTR promoter independently of the trans-activation response region. To show that the trans activation of the promoter by Tat directly involves the Sp1 protein, we have targeted a GAL-Sp1 fusion protein to the long terminal repeat promoter via upstream GAL4-binding sites. In the presence of Tat and GAL-Sp1, the promoter is synergistically trans activated at the transcriptional level, indicating that Tat and Sp1 functionally interact to trans activate the HIV-1 promoter. The Sp1 synergism is relatively specific, since another chimeric transcriptional activator, GAL-VP16, does not appear to be significantly synergistic with Tat.
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PMID:Synergistic activation of the human immunodeficiency virus type 1 promoter by the viral Tat protein and cellular transcription factor Sp1. 158 36

We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus E1a or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55% lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.
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PMID:The yeast GAL4 protein transactivates the polyomavirus origin of DNA replication in mouse cells. 164 81

As part of a study of transcriptional regulation by viral proteins, we examined whether an acidic region from a regulatory protein of an RNA virus could function as a trans-activator. The NH2-terminal highly acidic domain I of the phosphoprotein (P) of vesicular stomatitis virus (VSV) was fused to the DNA-binding domain of the yeast trans-activator, GAL4. In transient transfection assays, the resulting chimeric protein failed to activate transcription of a reporter CAT gene. However, mutation of basic amino acid residues located at positions 6 and 8 or the alteration of eight amino acids within the acidic domain to eight different amino acids converted the chimeric protein into a transcriptional activator comparable to wild-type GAL4. When subjected to SDS-polyacrylamide gel electrophoresis, the P proteins containing trans-activation-positive mutations in domain I showed an altered mobility, suggesting that these mutations may have caused a conformational change that is critical for trans-activation. Since the acidity of P domain I is not sufficient to activate transcription, additional features of this region must play an important role in GAL4-mediated trans-activation. None of the trans-activation-positive mutants supported VSV RNA transcription in vitro. These results suggest that the amino acid residues within P domain I that can be made to function in the trans-activation of DNA-dependent RNA transcription are distinct from those involved in VSV RNA-dependent RNA transcription.
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PMID:Alteration of specific amino acid residues in the acidic domain I of VSV phosphoprotein (P) converts a GAL4-P(I) hybrid into a transcriptional activator. 165 11

Epstein-Barr virus nuclear protein 2 (EBNA-2) increases mRNA levels of specific viral and cellular genes through direct or indirect effects on upstream regulatory elements. The EBNA-2 domains essential for these effects have been partially defined and correlate with domains important for B-cell growth transformation. To determine whether EBNA-2 has a direct transcriptional activating domain, gene fusions between the DNA-binding domain of GAL4 and EBNA-2 were tested in CHO and B-lymphoma cells for the ability to activate transcription from target plasmids containing GAL4 recognition sites upstream of an adenovirus or murine mammary tumor virus promoter. In B-lymphoma cells, a 37-amino-acid EBNA-2 domain previously identified to be essential for transformation was nearly as strong a transcriptional activator as the activating domain of herpes simplex virus trans-inducing factor VP16. A quadradecapeptide had about 25% of the activating activity of the longer peptide. This first evidence that EBNA-2 directly activates transcription should facilitate the identification of nuclear factors with which EBNA-2 interacts in transactivation and transformation.
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PMID:An Epstein-Barr virus nuclear protein 2 domain essential for transformation is a direct transcriptional activator. 165 76

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.
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PMID:Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative. 173 26

NGFI-A is a mammalian transcription factor that contains zinc fingers similar to those observed in several other proteins, including NGFI-C and Krox-20. To define precisely the DNA binding domain of NGFI-A, we selected mutants using a chimeric transcriptional activator that contains the NGFI-A zinc finger domain sandwiched between the lexA DNA binding domain and the GAL4 transcriptional activating domain. Expression of this lexA-NGFI-A-GAL4 (LAG) trimeric protein in yeast significantly retarded their growth, unlike an activator containing only the lexA and GAL4 components. This suggested that LAG inappropriately regulates genes in yeast that contain NGFI-A binding sites. Yeast that contained LAG reverted to wild-type growth at high frequency by inactivation of LAG. The mutations recovered from these revertants were specifically limited to the 83-residue NGFI-A zinc finger domain by requiring that the lexA and GAL4 portions of the LAG chimera remain functional. Nearly all of the 93 mutants obtained contained single missense mutations that mapped within the zinc fingers to residues thought to be important for zinc finger function. Deletion analysis of native NGFI-A verified that residues distant from the zinc fingers do not influence DNA binding, thus establishing the minimal functional DNA binding domain. Interestingly, many zinc finger residues ascribed specific functions by x-ray crystallography were never mutated in yeast, implying that the identity of these residues is not critical. Surprisingly, not all of the mutations tested significantly impaired NGFI-A-specific DNA binding, suggesting that the function of these zinc fingers is more diverse than previously recognized.
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PMID:In vivo mutational analysis of the NGFI-A zinc fingers. 174 Apr 23

Tax1 of human T-cell leukemia virus type 1 (HTLV-1) activates viral transcription dependent upon three 21-bp enhancer elements in the long terminal repeat. Difficulties in detecting any association of Tax1 with the viral enhancer have hampered elucidation of the molecular mechanisms of Tax1-mediated transcriptional activation. By constructing a fusion protein with the heterologous DNA-binding domain of yeast GAL4, Tax1 was shown to be a potent transcriptional activator dependent on the presence of GAL4-binding sites. Deletions of the Tax1 portion of the fusion protein revealed that almost the entire region of Tax1 (amino acids 2-337) is required for activation, and the activity correlated well with that of the viral enhancer. The GAL/Tax1 mutant lacking 41 residues of the C-terminus of Tax1, GAL/Tax1(2-312), was inactive for the viral enhancer, but activity was recovered by adding the heterologous activation domain of herpes simplex virus VP16. These results indicate that Tax1 has two distinct but overlapping functional domains for transcriptional activation and for enhancer specificity. Thus, Tax1 is thought to be a transcription factor acting in the enhancer complex rather than as a catalytic or allosteric modifier of pre-existing cellular transcription factors.
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PMID:HTLV-1 Tax has distinct but overlapping domains for transcriptional activation and for enhancer specificity. 176 79

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.
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PMID:Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements. 184 61


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