Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q02556 (DNA-binding domain)
 6,431 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.
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PMID:Role of GCR2 in transcriptional activation of yeast glycolytic genes. 150 87

Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein. GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain. Such proteins are thought to activate specific genes by complexing with DNA-bound proteins. To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11. The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S. cerevisiae) or 7% (K. lactis), and 8% proline (K. lactis) or 5% (S. cerevisiae) residues. Both proteins have runs of pure glutamines. Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues. The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K. lactis creates phenotypes similar to those seen previously in gal11-defective S. cerevisiae. In addition, Kl-GAL11 complements a gal11-defect in S. cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.
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PMID:Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains. 192 18

The GATA motif (WGATAR) is found in the promoter regions of numerous Caenorhabditis elegans genes, including two intestine-specific genes, vit-2 and ges-1, in which it has been shown to be required for promoter function. The protein ELT-1, encoded by a single-copy gene homologous to the GATA family of vertebrate transcription factors, is potentially capable of interacting with this element. In order to determine whether ELT-1 is a transcriptional activator that recognizes this sequence, we have expressed it under the control of the GAL1 promoter in yeast. lacZ driven by the CYC1 promoter lacking an upstream activation sequence (UAS) but containing GATA sequences was used as a reporter. beta-Galactosidase was expressed upon induction only when GATA sequences were present, and expression was increased dramatically by additional binding sites. Deletion analysis demonstrated that the C terminus, containing only one of the two zinc fingers, is sufficient for activation. In addition, the DNA-binding domain and two transactivation regions were identified by fusing these isolated domains to previously defined domains of heterologous transcription factors. While most single base alterations in the GATA core sequence eliminated activity, an A to C change in position four, creating a GATC core, was found to increase activity significantly. The deleted ELT-1 protein containing only the C-terminal Zn finger was sufficient for activation in response to GATA, but both fingers were required for activation at GATC. A variety of sites with non-optimal sequences surrounding the GATA core also were found to be excluded better by the protein containing both Zn fingers. Furthermore, a fusion protein containing the entire ELT-1 DNA binding domain fused to the VP16 activation domain was found to have an even greater preference for the GATC core, as well as the optimal flanking bases. We conclude that, although ELT-1 having only its C-terminal finger is capable of activation in response to the WGATAR site, the presence of the upstream finger supplies additional base specificity.
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PMID:Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast. 747 42

The peroxisome proliferator-activated receptor (PPAR) binds cooperatively to cognate peroxisome proliferator-responsive elements (PPRE) in vitro through heterodimerization with retinoid X receptors (RXR). We used the yeast two-hybrid system to determine whether these two nuclear receptors physically interact in vivo. Mouse (m) PPAR and human (h) RXR alpha were synthesized as fusion proteins to either the DNA-binding domain (GBD) or the transactivation domain (GAD) of the yeast GAL4 transcription-activator protein, and were tested for their ability to activate expression of a GAL1::lacZ reporter gene. Strong activation was observed only in yeast transformed with combinations of GBD::mPPAR and GAD::hRXR alpha or with GAD::mPPAR and GBD::hRXR alpha. Homodimeric interaction by mPPAR was not detected. These results provide evidence for the interaction of PPAR and RXR alpha in vivo in the absence of a PPRE target site or exogenously added ligands.
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PMID:The peroxisome proliferator-activated receptor interacts with the retinoid X receptor in vivo. 795 63

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.
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PMID:Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI. 841 50

Transcription of the genes required for utilization of galactose in Saccharomyces cerevisiae is controlled primarily by the transcriptional activator protein GAL4. The upstream activating sequences for galactose (UASG) of most GAL genes have multiple sites to which GAL4 can bind. In this report we compare the binding properties of wild type GAL4 and derivatives of GAL4 bearing the N-terminal DNA-binding domain to multiple DNA-binding sites in vitro. To produce wild type GAL4, we constructed a recombinant baculovirus for expression in insect cells. Recombinant wild type GAL4 was found to bind efficiently to an oligonucleotide containing a near-consensus 17-mer GAL4 DNA-binding site in electrophoretic mobility shift assays. Footprinting experiments revealed that wild type GAL4 binds cooperatively to the four GAL4 DNA-binding sites of the GAL1-10 UASG; however, in contrast an N-terminal fragment of GAL4 containing only the DNA-binding/dimerization domains binds to each of these sites with slightly different affinity. With increasing concentrations of GAL4(1-147), the four sites become filled in the following order: site II, site IV, site I, and site III. In experiments with wild type GAL4, these four sites become fully occupied at approximately the same concentration of protein. In footprints of wild type GAL4 on the USAG, enhancements and protections of DNase I-sensitive cleavages are detectable between sites III and IV, indicative of formation of a loop between these distantly spaced sites. Binding of wild type GAL4 to a strong near-consensus binding site assists binding to an adjacent mutant site in both electrophoretic mobility shift and footprinting assays. GAL4(1-147) and GAL4(1-147) fused to portions of GAL4's activating region II were incapable of cooperative DNA binding in our assays. We conclude from these observations that wild type GAL4 has a cooperative DNA-binding function that is distinct from the DNA binding and dimerization or transcriptional activation functions, and likely plays and important role in precise regulation of GAL gene transcription.
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PMID:Wild type GAL4 binds cooperatively to the GAL1-10 UASG in vitro. 848 50

This report describes a novel yeast one-hybrid system which easily allows for the detection of mutations in the ligand-binding domain of the estrogen receptor. This screen is based on the observation that a fusion protein consisting of the GAL4 DNA-binding domain and the estrogen receptor can interact with a GAL4 upstream activating sequence and induce the expression of an integrated GAL1-lacZ gene only in the presence of estradiol. Various deletion mutants of the estrogen receptor were tested in this assay and activating function 1 which is present in the N-terminus of the estrogen receptor was found to be responsible for the transactivation produced in the assay. To test if the screen could be used to detect random mutants in the ligand-binding domain of the estrogen receptor the region of the human receptor between amino acids 381 to 403 was mutated by oligonucleotide saturation mutagenesis. Two of the mutants generated by this mutagenesis were characterized to demonstrate that the results obtained from the screen in the yeast screen are relevant to mammalian systems. One of the mutants which has a valine at position number 388 instead of a glycine was able to transactivate in both the yeast and a mammalian system. This mutant was a more potent activator of transcription and also appeared to have a higher affinity for [3H]estradiol in vivo than the wild type receptor. The other mutant which was characterized has five amino acid changes from amino acids 390 through 400. This mutant was nonfunctional in the yeast and mammalian transcription assays and did not bind [3H]estradiol in vivo or in vitro.
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PMID:Use of the yeast one-hybrid system to screen for mutations in the ligand-binding domain of the estrogen receptor. 885 26

mms4-1 is one of several Saccharomyces cerevisiae mutants that exhibit an increased sensitivity to methyl methanesulfonate (MMS), but not to UV or X-rays. We have isolated the MMS4 gene by functional complementation of the MMS-sensitive phenotype in the mms4-1 strain. The MMS4 gene encodes a 691-amino acid, 78.7-kDa protein. The deduced Mms4 protein does not show significant homology to any of the known proteins in the database. However, several putative functional domains suggest that it may be a nuclear protein capable of interacting with other proteins. Examination of the mms4delta mutant phenotype indicates that the mutation not only sensitizes DNA to methylating and ethylating agents, but also to other DNA damage that blocks DNA replication. However, the mms4delta mutant appears to be more sensitive to chronic treatment than to acute treatment by DNA-damaging agents. Furthermore, the spontaneous mutation rate increases significantly in the mms4delta mutant. Mms4 alone, when fused to a Gal4 DNA-binding domain, is able to activate P(GAL1)-lacZ and P(GAL1)-HIS3 reporter genes in a two-hybrid system; the Mms4 transactivation domain maps to the highly acidic N-terminal region. These results collectively suggest that Mms4 may function as a transcriptional (co)activator and play an important role in DNA repair and/or synthesis.
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PMID:Mms4, a putative transcriptional (co)activator, protects Saccharomyces cerevisiae cells from endogenous and environmental DNA damage. 960 84

The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (-533 to -544) which was characterized as an upstream repressing sequence (URSMIG-ENA1) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at -502 to -513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URSCRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase-Sko1 fusion protein binds specifically to the URSCRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URSCRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URSCRE-ENA1 and URSMIG-ENA1) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or ssn6 mutants to Na+ or Li+ stress.
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PMID:Repressors and upstream repressing sequences of the stress-regulated ENA1 gene in Saccharomyces cerevisiae: bZIP protein Sko1p confers HOG-dependent osmotic regulation. 985 77

To determine whether similar regulatory mechanisms control the expression of glycolytic genes in yeast and human cells, we screened a human brain cDNA library for clones which complement the growth defect of the gcr2 mutant of Saccharomyces cerevisiae, and isolated hSGT1 (human suppressor of GCR two). Further work confirmed that the rescue of growth was associated with recovery of glycolytic enzyme activities, and that hSGT1 did not complement the growth defect of a gcr1 mutant. A hybrid protein comprising hSgt1p and the DNA-binding domain of Gal4p (GBD) activated a GAL1-lacZ reporter gene fusion, suggesting that the cloned gene may be a transcriptional activator. Two-hybrid experiments in yeast also indicate that hSgt1p interacts with Gcr1p. Northern analysis showed that hSGT1 is highly expressed in muscle and heart. Although the predicted amino acid sequence of hSgt1p does not display significant similarity to Gcr2p, we speculate that their functions may be analogous.
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PMID:A human gene, hSGT1, can substitute for GCR2, which encodes a general regulatory factor of glycolytic gene expression in Saccharomyces cerevisiae. 992 32


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