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

We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases. The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4. The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding. Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C . G base pair. Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent. The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA.
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PMID:Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein. 267 75

The transcription of the 14 gvp genes of the gas-vesicle-encoding mc-vac region was investigated, using RNA from 25% and 15% (w/v) salt cultures of the moderately halophilic archaeon Haloferax mediterranei. Transcription occurred only from two promoters, located in front of the mc-gvpA and mc-gvpD genes. In both cultures transcripts spanning the entire mc-gvpDEFGHIJKLM transcription unit were formed only during the exponential growth phase. Amounts of these transcripts were larger in the 25% salt culture, in which the 2.0 kb mc-gvpD transcripts were also synthesized during the stationary phase. The levels of the mc-gvpD transcripts and of the 324 nt mc-gvpA mRNA increased in parallel during the stationary phase of the 25% salt culture. Only under these conditions were mRNAs spanning the entire mc-gvpACNO transcription unit observed, and gas-vesicles were formed. Investigation of the influence of the mc-gvpDE genes on both mc-vac promoters in transformants revealed that by themselves they were nearly inactive. The addition of mc-gvpE, however, resulted in a high level of constitutively produced mc-gvpA and mc-gvpD mRNA, indicating a transcriptional activator function for the mc-gvpE product.
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PMID:Influence of salt on the transcription of the gas-vesicle genes of Haloferax mediterranei and identification of the endogenous transcriptional activator gene. 875 36

Yeast cells deficient in the transcriptional activator Imp2p are viable, but display marked hypersensitivity to a variety of oxidative agents. We now report that imp2 null mutants are also extremely sensitive to elevated levels of the monovalent ions, Na+ and Li+, as well as to the divalent ions Ca2+, Mn2+, Zn2+, and Cu2+, but not to Cd2+, Mg2+, Co2+, Ni2+, and Fe2+, as compared to the parent strain. We next searched for multicopy suppressor genes that would allow the imp2Delta mutant to grow under high salt conditions. Two genes that independently restored normal salt-resistance to the imp2Delta mutant, ENA1 and HAL3, were isolated. ENA1 encodes a P-type ion pump involved in monovalent ion efflux from the cell, while HAL3 encodes a protein required for activating the expression of Ena1p. Neither ENA1 nor HAL3 gene expression was positively regulated by Imp2p. Moreover, the imp2 ena1 double mutant was exquisitely sensitive to Na+/Li+ cations, as compared to either single mutant, implying that Imp2p mediates Na+/Li+ cation homeostasis independently of Ena1p.
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PMID:The transcriptional activator Imp2p maintains ion homeostasis in Saccharomyces cerevisiae. 961 Dec

In the transcriptional response of Saccharomyces cerevisiae to stress, both activators and repressors are implicated. Here we demonstrate that the ion homeostasis determinant, HAL1, is regulated by two antagonistically operating bZIP transcription factors, the Sko1p repressor and the Gcn4p activator. A single CRE-like sequence (CRE(HAL1)) at position -222 to -215 with the palindromic core sequence TTACGTAA is essential for stress-induced expression of HAL1. Down-regulation of HAL1 under normal growth conditions requires specific binding of Sko1p to CRE(HAL1) and the corepressor gene SSN6. Release from this repression depends on the function of the high-osmolarity glycerol pathway. The Gcn4p transcriptional activator binds in vitro to the same CRE(HAL1) and is necessary for up-regulated HAL1 expression in vivo, indicating a dual control mechanism by a repressor-activator pair occupying the same promoter target sequence. gcn4 mutants display a strong sensitivity to elevated K(+) or Na(+) concentrations in the growth medium. In addition to reduced HAL1 expression, this sensitivity is explained by the fact that amino acid uptake is drastically impaired by high Na(+) and K(+) concentrations in wild-type yeast cells. The reduced amino acid biosynthesis of gcn4 mutants would result in amino acid deprivation. Together with the induction of HAL1 by amino acid starvation, these results suggest that salt stress and amino acid availability are physiologically interconnected.
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PMID:The Sko1p repressor and Gcn4p activator antagonistically modulate stress-regulated transcription in Saccharomyces cerevisiae. 1111 77

From Ralstonia eutropha HF39 null-allele mutants were created by Tn5 mutagenesis and by homologous recombination which were impaired in growth on propionic acid and levulinic acid. From the molecular, physiological and enzymic analysis of these mutants it was concluded that in this bacterium propionic acid is metabolized via the methylcitric acid pathway. The genes encoding enzymes of this pathway are organized in a cluster in the order prpR, prpB, prpC, acnM, ORF5 and prpD, with prpR transcribed divergently from the other genes. (i) prpC encodes a 2-methylcitric acid synthase (42720 Da) as shown by the measurement of the respective enzyme activity, complementation of a prpC mutant of Salmonella enterica serovar Typhimurium and high sequence similarity. (ii) For the translational product of acnM the function of a 2-methyl-cis-aconitic acid hydratase (94726 Da) is proposed. This protein and also the ORF5 translational product are essential for growth on propionic acid, as revealed by the propionic-acid-negative phenotype of Tn5-insertion mutants, and are required for the conversion of 2-methylcitric acid into 2-methylisocitric acid as shown by the accumulation of the latter, which could be purified as its calcium salt from the supernatants of these mutants. In contrast, inactivation of prpD did not block the ability of the cell to use propionic acid as carbon and energy source, as shown by the propionic acid phenotype of a null-allele mutant. It is therefore unlikely that prpD from R. eutropha encodes a 2-methyl-cis-aconitic acid dehydratase as proposed recently for the homologous prpD gene from S. enterica. (iii) The translational product of prpB encodes 2-methylisocitric acid lyase (32314 Da) as revealed by measurement of the respective enzyme activity and by demonstrating accumulation of methylisocitric acid in the supernatant of a prpB null-allele mutant. (iv) The expression of prpC and probably also of the other enzymes is regulated and is induced during cultivation on propionic acid or levulinic acid. The putative translational product of prpR (70895 Da) exhibited high similarities to PrpR of Escherichia coli and S. enterica, and might represent a transcriptional activator of the sigma-54 family involved in the regulation of the other prp genes. Since the prp locus of R. eutropha was very different from those of E. coli and S. enterica, an extensive comparison of prp loci available from databases and literature was done, revealing two different classes of prp loci.
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PMID:The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism. 1149 97

We report that RapA, an Escherichia coli RNA polymerase (RNAP)-associated homolog of SWI2/SNF2, is capable of dramatic activation of RNA synthesis. The RapA-mediated transcriptional activation in vitro depends on supercoiled DNA and high salt concentrations, a condition that is likely to render the DNA superhelix tightly compacted. Moreover, RapA activates transcription by stimulating RNAP recycling. Mutational analyses indicate that the ATPase activity of RapA is essential for its function as a transcriptional activator, and a rapA null mutant exhibits a growth defect on nutrient plates containing high salt concentrations in vivo. Thus, RapA acts as a general transcription factor and an integral component of the transcription machinery. The mode of action of RapA in remodeling posttranscription or posttermination complexes is discussed.
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PMID:RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA polymerase recycling in transcription. 1175 38

Four orthologues of the Arabidopsis CBF/Dreb transcriptional activator genes were isolated from the winter Brassica napus, cv. Jet neuf. All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences suggested that BNCBFs 5, 7and 16 are very similar to the Arabidopsis CBFI whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain. Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the Brassica seedlings and preceded transcript accumulation of the cold-inducible BN28 gene, a Brassica orthologue of the cor6.6 or KIN gene from Arabidopsis. Cold-induced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16. Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress. BNCBF17 was not responsive to salt stress. BNCBF transcript accumulation was similar in both spring and winter Brassica but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type. BNCBF5 and 17 proteins bind in vitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters. Differential binding preferences, however, to LTREs between BNI 15 and BN28 were observed. Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5. Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17 trans-activation of the Lac-Z reporter gene in yeast. We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in Brassica, controlling the expression of cold-induced genes through an ABA-independent pathway.
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PMID:Regulation and characterization of four CBF transcription factors from Brassica napus. 1209 Jun 22

Apetala2/ethylene-responsive factor (AP2/ERF) proteins are AP2 domain-containing transcription factors and form the second largest transcription factor family in plants. Biological functions of many of these AP2 proteins are still unknown. Here, we report the characterisation of a novel member of the AP2/ERF superfamily, dehydration-responsive factor 1 (HvDRF1) from barley, and its role in abscisic acid (ABA)-mediated gene regulation. The expression of HvDRF1 was upregulated in barley leaves and roots under drought, salt or ABA treatment, and in embryos during seed maturation. Three forms of HvDRF1 transcripts were produced through alternative splicing, two of which encoded AP2 proteins. This alternative splicing pattern was also observed in a wheat homologue gene, TaDRF1. Both of HvDRF1 AP2 proteins acted as transcriptional activators, capable of activating the promoter activity of an ABA-inducible HVA1s in barley. In vitro DNA-binding analysis using synthetic oligonucleotides revealed that HvDRF1 AP2 protein bound preferably to a CT-rich element (T(T/A)ACCGCCTT). HvDRF1 activity on the activation of HVA1s expression in barley leaves was markedly enhanced by HvABI5 (a bZIP transcription factor), ABA or drought treatment. These results indicate that the HvDRF1 transcriptional activator co-operates with other ABA-responsive factors in the upregulation of stress gene expression through an ABA-dependent pathway.
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PMID:HvDRF1 is involved in abscisic acid-mediated gene regulation in barley and produces two forms of AP2 transcriptional activators, interacting preferably with a CT-rich element. 1473 Dec 54

In the yeast Saccharomyces cerevisiae, starvation for amino acids induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2alpha by Gcn2 protein kinase, leading to elevated translation of GCN4. Gcn4p is a transcriptional activator of hundreds of genes involved in remedying nutrient deprivation. In addition to a conserved kinase domain, Gcn2p has a regulatory region homologous to histidyl tRNA synthetase enzymes that binds uncharged tRNA that accumulates during amino acid starvation. Flanking the carboxyl terminus of the histidyl-tRNA synthetase-related domain is a region spanning 162 residues that participates in the activation of the protein kinase. Gel filtration and chemical cross-linking analysis of the recombinant carboxyl-terminal Gcn2 protein revealed that this region is a stable homodimer that is highly resistant to high concentrations of salt. Residue alterations in three hydrophobic segments and one segment with a proposed amphipathic alpha-helix in this Gcn2p carboxyl terminus blocked oligomerization, supporting the role of hydrophobic interactions in the dimerization interface of Gcn2p. Introduction of residue substitutions that impaired dimerization into the full-length protein prevented the ability of Gcn2p to phosphorylate its substrate eukaryotic initiation factor-2alpha and induce GCN4 translational expression in yeast cells subjected to a variety of stresses including amino acid limitation or exposure to rapamycin or high levels of NaCl. This latter stress can be overcome by addition of increasing amounts of K+ ions, indicating that the Na+/K+ ion balance is central to this stress induction. We conclude that dimerization involving hydrophobic segments in the carboxyl-terminal region is required for activation of Gcn2p in response to a multitude of stresses.
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PMID:Dimerization is required for activation of eIF2 kinase Gcn2 in response to diverse environmental stress conditions. 1501 Apr 61

Double mutant cycle analysis was employed to ascertain the role of intra- and interchain salt-bridges in the folding and stability of the dimeric coiled-coil peptide, GCN4-p1, the 33-residue leucine zipper domain of the transcriptional activator GCN4. Equilibrium circular dichroism studies of the urea-induced unfolding reaction at neutral pH revealed that both types of ionic interactions, localized primarily in the N-terminal portion of the molecule, enhance the stability of the native coiled-coil. By contrast, comparable stopped-flow circular dichroism studies indicate that the salt-bridge interactions, with one possible exception, are not well formed in the transition state for folding. Although the E22Q/R25A double mutant failed to fold, fragmentation studies suggest that the E22/R25 intramolecular salt-bridge may play a critical role in stabilizing C-terminal nascent helices that drive the association reaction. The remaining salt-bridges appear to stabilize the parallel-stranded coiled-coil architecture of GCN4-p1 only after the peptide traverses the rate-limiting, dimeric transition state.
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PMID:Salt-bridges can stabilize but do not accelerate the folding of the homodimeric coiled-coil peptide GCN4-p1. 1503 63


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