Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.
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PMID:The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding. 144 98

The transcriptional mechanisms which contribute to the regulation of nerve growth factor (NGF) production are still largely unknown. We previously expressed the NGF promoter region in transgenic mice to localize cis regulatory elements to within 5 kb of the promoter. To further map these elements, and to begin to study the corresponding transacting factors, we here assayed the effects of 5' deletions and point mutations and examined the binding of nuclear factors to the NGF promoter region using L929 cell fibroblasts. Sequential deletions delineated regions upstream from the promoter which stimulated and inhibited transcription. DNAse-1 footprinting experiments identified four upstream segments, designated F2, F4, F6 and F8, which bound L929 cell nuclear proteins. F2 and F4 mapped to stimulatory and F6 and F8 to inhibitory regions. Competition experiments using a heptanucleotide present in both F2 and F4 segments suggested that they may be bound by related factors. Gel shift assays showed that the F8 binding proteins are less abundant in L929 cells than in NIH 3T3 fibroblasts and B16 melanoma cells. In addition to the upstream segments, a downstream AP-1 consensus sequence bound L929 nuclear proteins. Mutation of the AP-1 consensus sequence eliminated binding of nuclear proteins and reduced transcriptional activity. Our results indicate that transcriptional activator as well as suppressor regions surround the NGF gene promoter. The regulation of NGF production is likely to involve cis elements within these regions and transacting factors that bind to them.
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PMID:Structural and functional identification of regulatory regions and cis elements surrounding the nerve growth factor gene promoter. 166 23

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

Transcription of numerous virulence genes in Bordetella pertussis is positively regulated by the products of the bvgAS genes. In this study a series of lacZYA fusions containing deletions in either the fhaB or bvgA promoter regions was used to identify cis-acting regulatory regions required for bvg activation of these two genes. Gel retardation and DNase I protection analyses have shown that specific protein-DNA interactions occur at these regulatory regions and that these interactions require the transcriptional activator protein BvgA. The regulatory regions found upstream of fhaB and bvgA which are involved in protein binding both contain the sequence TTTCCTA. This sequence is part of an inverted repeat upstream of fhaB and a direct repeat upstream of bvgA. Homologous repeats are not apparent upstream of other bvg-activated genes, such as ptx and cyaA. These data suggest that the mechanism for transcriptional regulation of the other bvg-activated genes is complex and may require regulatory factors in addition to the bvgAS gene products.
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PMID:Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon. 200 57

Endothelin-1 (ET-1) is a 21-amino-acid peptide synthesized by endothelial cells that has potent vasoconstrictor activity. Human ET-1 is derived from a 212-amino-acid prepropeptide, termed preproendothelin-1 (PPET-1). To identify cis-acting sequences essential for PPET-1 gene transcription, bovine aortic endothelial (BAE) cells were transfected with plasmids containing 5'-flanking sequences of the human PPET-1 gene fused to the human growth hormone gene as a reporter. Deletional analysis of these fusion plasmids showed that the sequence spanning positions -141 to -127 of the human PPET-1 promoter is required for full transcription activity. Introduction of clustered point mutations into this region of the promoter reduced transcription activity. Gel shift analysis, methylation interference, protein-DNA cross-linking, and oligonucleotide competition studies revealed that BAE cell nuclear extract contains a 47-kilodalton DNA-binding protein recognizing the core motif TATC (GATA) located at positions -135 to -132 of the PPET-1 promoter. The size and specificity of this DNA-binding protein resemble GF-1, a previously described transcription factor of erythroid cells that binds to the same core motif. Gel shift analysis indicated that GF-1 and the DNA-binding protein interacting with the PPET-1 promoter have different tissue distributions; the former is restricted to a subset of hematopoietic cells, and the latter is found in various cell types, including BAE, NIH 3T3, and HeLa cells. By using an antiserum to the C-terminal region of GF-1, the two proteins were also found to be antigenically distinct. When a growth hormone fusion plasmid containing the proximal 141 nucleotides of the PPET-1 promoter was transfected into a variety of cell types, these was preferential expression in cells of endothelial origin. We conclude that a nuclear factor with binding specificity for a GATA motif similar to that of the transcriptional activator GF-1 is necessary for the efficient and cell-specific expression of the human PPET-1 gene.
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PMID:A nonerythroid GATA-binding protein is required for function of the human preproendothelin-1 promoter in endothelial cells. 238 28

The DNA context of nucleotides that a protein recognizes can influence the strength of the protein-DNA interaction. Moreover, in prokaryotes, understanding the quantitative differences in binding affinities that result in part from the DNA context is often important in describing regulatory mechanisms. Nevertheless, these issues have not been a major focus yet for the investigation of protein-DNA interactions in eukaryotes. In this study, we explored the binding specificity and the range of affinities that the BPV-1 E2 transcriptional activator has for DNA. Because E2 binding sites are positioned near several different BPV-1 promoters, such quantitative information may be important to understand transcriptional regulatory mechanisms in BPV-1. Gel retardation assays and DNA footprinting were used to quantitate the affinities of the E2 binding sites in the viral genome. In the process, five sites were discovered, which, on the basis of sequence, had not been predicted previously to interact with the E2 protein. Equilibrium and kinetic studies show that the range of E2 affinities of the 17 sites varied over 300-fold. The sequence elements responsible for E2 recognition of DNA were determined by missing contact analysis of several sites and a point mutation analysis of one site. The results presented show that the affinity of an E2 binding site is to a large extent determined by the availability of specific contacts, but the data also strongly suggest that DNA structure plays an important role.
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PMID:Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome. 254 29

On the basis of the constitutive phenotypes of two catM mutants of Acinetobacter calcoaceticus, the CatM protein was proposed to repress expression of two different loci involved in catechol degradation, catA and catBCIJFD (E. Neidle, C. Hartnett, and L. N. Ornston, J. Bacteriol. 171:5410-5421, 1989). In spite of its proposed negative role as a repressor, CatM is similar in amino acid sequence to positive transcriptional activators of the LysR family. Investigating this anomaly, we found that insertional inactivation of catM did not cause the phenotype expected for the disruption of a repressor-encoding gene: in an interposon-generated catM mutant, no cat genes were expressed constitutively, but rather catA and catB were still inducible by muconate. Moreover, this catM mutant grew poorly on benzoate, a process requiring the expression of all cat genes. The inducibility of the cat genes in this catM mutant was completely eliminated by a 3.5-kbp deletion 10 kbp upstream of catM. In this double mutant, catM in trans restored muconate inducibility to both catA and catB. These results suggested the presence of an additional regulatory locus controlling cat gene expression. The ability of CatM to function as an activator was also suggested by these results. In support of this hypothesis, in vivo methylation protection assays showed that CatM protects two guanines in a dyad 65 nucleotides upstream of the catB transcriptional start site, in a location and pattern typical of LysR-type transcriptional activators. Gel mobility shift assays indicated that CatM also binds to a region upstream of catA. DNA sequence analysis revealed a nucleotide near the 3' end of catM not present in the published sequence. Translation of the corrected sequence resulted in the deduced CatM protein being 52 residues longer than previously reported. The size, amino acid sequence, and mode of action of CatM now appear similar to, and typical of, what has been found for transcriptional activators in the LysR family. Analysis of one of the constitutive alleles of catM previously thought to encode a dysfunctional repressor indicated instead that it encodes an inducer-independent transcriptional activator.
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PMID:catM encodes a LysR-type transcriptional activator regulating catechol degradation in Acinetobacter calcoaceticus. 759 40

The PEA3 group is a homogeneous group of the ets transcription factor family and is composed of three known members, PEA3, ERM and ER81, which, on the amino acid (AA) level, are more than 95% identical within the DNA-binding domain (the Ets domain), more than 85% within a 32 AA domain (the acidic domain) localized in the amino-terminus and almost 50% identical overall. By screening a human kidney cDNA library with a specific probe obtained from mouse ER81, we isolated two clones of 1.6 and 1.5 kb in length encoding a 458 AA open reading frame. When compared to mouse ER81, the present human ER81 lacks the last 13 AA of the acidic domain and the 5 AA contiguous to the carboxy-terminal part of the acidic domain. Of the 458 AA of the human ER81 protein, 97% are identical to mouse ER81. Gel shift analysis indicates that the full-length human ER81 protein is able to bind specifically to an oligonucleotide containing the binding sites recognized by most of the Ets proteins. By transient expression in RK13 mammalian cells, human ER81 protein transactivated a reporter plasmid containing Ets binding sites, indicating that this molecule is a bonafide transcriptional activator, while by expression in Cos-1 transfected cells, we detected the presence of human ER81 protein in the nucleus using immunocytochemistry. In human tissues, ER81 mRNA is very highly expressed in brain, highly expressed in testis, lung and heart, moderately in spleen, small intestine, pancreas and colon, weakly in liver, prostate and thymus, very weakly in skeletal muscle, kidney and ovary and not in placenta and peripheral blood leukocytes. Analysis of human solid or haematopoietic tumour cell lines showed that most of them did not express ER81 detectably. Database searches revealed that ETV1 mRNA is highly similar to human ER81 described here, although it contains the full-length acidic domain present in mouse ER81. By screening a genomic DNA library, we characterized the intron-exon junction within the acidic domain of human ER81 and we showed that this junction corresponds to the site where the remaining AA of the acidic domain of ETV1 or mouse ER81 are inserted.
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PMID:Molecular characterization of the ets-related human transcription factor ER81. 765 41

The uaY gene codes for a transcriptional activator mediating the induction of a number of unlinked genes involved in purine utilization in Aspergillus nidulans. Here we present the complete genomic and cDNA nucleotide sequence of this gene. The gene contains two introns. The derived polypeptide of 1060 residues contains a typical zinc binuclear cluster domain and shows a number of similarities with the PPR1 regulatory gene of Saccharomyces cerevisiae. These similarities are most striking in the putative linker and dimerization regions following the zinc cluster. Gel-shift and DNase I footprinting experiments have been carried out for three genes subject to UaY-mediated induction. The binding sequence is 5'-TCGG-6X-CCGA, which is identical to the proposed PPR1 binding sites. Nevertheless, the identity of the base immediately 3' of the 5'-TCGG sequence clearly affects the affinity of the site. The site upstream of the uapA gene has been shown to be active in vivo. Binding to this site has been analysed by a number of interference techniques. There is an interesting chemical similarity between the co-inducer of the purine utilization pathway (uric acid) and that of the genes of the pyrimidine biosynthetic pathway (dihydroorotic acid) and we show that dihydroorotic acid can act as a poor inducer of at least one activity under UaY control. These striking similarities, together with the unique pattern of regulation of pyrimidine biosynthesis in S. cerevisiae, suggest that PPR1 evolved through recruitment into the pyrimidine biosynthetic pathway of an ancestral gene related to uaY.
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PMID:The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae. 772 21

The major regulator of papillomavirus transcription is encoded by the viral E2 gene. The E2 gene has been well characterized in bovine papillomavirus (BPV) where it encodes at least three different polypeptides which differentially affect viral gene expression. In human papillomaviruses (HPVs) the E2 gene product is much less well characterized. In this study we have analysed the mechanism of action of the HPV-16, HPV-18 and BPV-1 E2 proteins in cervical keratinocytes. We show that the full length HPV E2 protein acts as a potent transcriptional activator of viral gene expression in both normal and immortalized keratinocytes. In contrast, the BPV-1 E2 protein produces transcriptional repression under identical conditions. A cDNA encoding the C-terminal half of the HPV-16 E2 protein in these assays weakly repressed viral gene expression. Further, co-transfection of this cDNA with the full length clone progressively abolishes the activation in trans by the full length HPV E2 protein. Gel retardation assays have defined a number of protein complexes between the long and short forms of E2 but with no evidence for preferential DNA binding. These results define two distinct activities for the HPV-16 E2 protein, indicate functional differences with the BPV E2 protein and suggest that splicing of the HPV E2 mRNA is a critical mechanism for controlling viral gene expression.
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PMID:Characterization of the human papillomavirus E2 protein: evidence of trans-activation and trans-repression in cervical keratinocytes. 795 11


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