UNIPROT:P51532 - FACTA Search

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

We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21. In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro. We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity. Binding of inducer [Hg(II)] to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD. Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription. Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD. Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay.
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PMID:A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. 133 70

Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a-r-) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a-r+) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variably deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data, taken with the results in the preceding paper (Shewchuk et al., 1989), support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation.
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PMID:Transcriptional switching by the MerR protein: activation and repression mutants implicate distinct DNA and mercury(II) binding domains. 249 78

The NS1 polypeptide of minute virus of mice (MVM) is a potent transcriptional activator of the MVM P38 promoter. The minimum region of this promoter required for transactivation has been identified and termed the transactivation region (tar). However, the function of tar and the biochemical steps involved in NS1-mediated transactivation are not well understood. Here we provide evidence that NS1 binds directly and specifically to tar in a strictly ATP-dependent manner. A DNA fragment containing tar was specifically coimmunoprecipitated with purified baculovirus-expressed MVM NS1, using antibodies directed against NS1 amino- or carboxy-terminal peptides. Using this immunoprecipitation assay, we found that the NS1-tar interaction was enhanced approximately 10-fold by ATP, but subsequent incubation at elevated temperatures in the presence, but not the absence, of MgCl2 caused rapid loss of tar binding. This finding suggests that the tar-NS1 complex has a short half-life under assay conditions which favor ATP hydrolysis. Specific binding was efficiently inhibited by self-ligated oligonucleotides containing the core DNA sequence (ACCA)3, but the same nonligated 20- and 21-mer oligonucleotides were unable to compete effectively, indicating that NS1 only binds to its cognate site when this site is presented on DNA fragments of sufficient size. DNase I footprinting experiments performed in the presence of gamma S-ATP revealed that NS1 protects a 43-bp sequence extending asymmetrically from the (ACCA)2 sequence toward the TATA box of the promoter. NS1 footprints obtained at other sites in the MVM genome were similarly large and asymmetric, all extending approximately 31 bp 5' from the core (ACCA)2-3 sequence. Surprisingly, no footprints were obtained in the absence of gamma S-ATP even under low-stringency binding conditions. However, ATP could be omitted from the reactions if NS1 was first incubated with antibodies directed against its 16-amino-acid carboxy-terminal peptide. Since these antibodies probably create intermolecular cross-links, this finding suggests that NS1 may only bind its cognate site efficiently, or perhaps at all, if the transactivator is first induced to form oligomers. From these data, we hypothesize that ATP binding may also induce NS1 to oligomerize and that such assembly is required before the protein can bind effectively to the tar sequence. The functional implications of the NS1-tar interaction will be discussed.
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PMID:Minute virus of mice transcriptional activator protein NS1 binds directly to the transactivation region of the viral P38 promoter in a strictly ATP-dependent manner. 763 87

The EBNA-2 protein is essential for the establishment of a latent Epstein-Barr virus (EBV) infection and for B-cell immortalization. EBNA-2 functions as a transcriptional activator that modulates viral latency gene expression as well as the expression of cellular genes, including CD23. We recently demonstrated that EBNA-2 transactivation of the EBV latency C promoter (Cp) is dependent on an interaction with a cellular DNA-binding protein, CBF1, for promoter targeting. To determine whether targeting via CBF1 is a common mechanism for EBNA-2-mediated transactivation, we have examined the requirements for activation of the cellular CD23 promoter. Binding of CBF1 to a 192-bp mapped EBNA-2-responsive region located at position -85 bp to -277 bp upstream of the CD23 promoter was detected in electrophoretic mobility shift assays. The identity of the bound protein as CBF1 was established by showing that the bound complex was competed for by the CBF1 binding site from the EBV Cp, that the bound protein could be supershifted with a bacterially expressed fusion protein' containing amino acids 252 to 425 of EBNA-2 but was unable to interact with a non-CBF1-binding EBNA-2 mutant (WW323SR), and that in UV cross-linking experiments, the Cp CBF1 binding site and the CD23 probe bound proteins of the same size. The requirement for interaction with CBF1 was demonstrated in a transient cotransfection assay in which the multimerized 192-bp CD23 response region was transactivated by wild-type EBNA-2 but not by the WW323SR mutant. Reporter constructions carrying multimerized copies of the 192-bp CD23 response region or multimers of the CBF1 binding site from the CD23 promoter were significantly less responsive to EBNA-2 transactivation than equivalent constructions carrying a multimerized region from the Cp or multimers of the CBF1 binding site from the Cp. Direct binding and competition assays using 30-mer oligonucleotide probes representing the individual CBF1 binding sites indicated that CBF1 bound less efficiently to the CD23 promoter and the EBV LMP-1 promoter sites than to the Cp site. To investigate the basis for this difference, we synthesized a series of oligonucleotides carrying mutations across the CBF1 binding site and used these as competitors in electrophoretic mobility shift assays. The competition experiments indicated that a central core sequence, GTGGGAA, common to all known EBNA-2-responsive elements, is crucial for CBF1 binding. Flanking sequences on either side of this core influence the affinity for CBF1.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:EBNA-2 upregulation of Epstein-Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1. 805 21

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

To facilitate the understanding of the complex process of target gene expression and its control, we report a modified inducible system for activation or repression of target gene expression in response to an exogenously administered compound. The main component of this inducible system is a chimeric transcriptional activator (GLVP) consisting of an N-terminal VP16 transcriptional activation domain fused to a yeast GAL4 DNA binding domain and a mutated human progesterone receptor (hPR) ligand binding domain (LBD). This chimeric regulator binds to a target gene containing the 17-mer GAL4 upstream activation sequence (UAS) in the presence of anti-progesterone, RU486. We showed that the combination of two different types of domains (VP16 and poly-glutamine stretch) into one chimeric molecule could result in a further increase in transcriptional activation potency. Through mutational analysis, we modified the original GLVP and generated a more potent version of the RU486 inducible regulator GL914 VPc with a 19 amino acid deletion of the hPR-LBD (delta C19) and a C-terminally located VP16 activation domain. More importantly, this new chimeric regulator can effectively activate target gene expression at a much lower concentration of RU486 (0.01 nM). The concept of RU486 regulatable gene expression is not limited to gene activation. By replacing the VP16 activation domain with a KRAB transcriptional repression domain, we are able to achieve inducible repression of target gene expression. We also present evidence that individual functional domains within a chimeric protein could modulate each other's function depending on their relative positions within the molecule. Using this potent regulator, we demonstrate that inducible nerve growth factor (NGF) secretion into conditioned media can elicit neurite outgrowth in co-cultured PC12 cells. This new versatile inducible system can potentially be used to control target gene expression in a mammalian system in vivo.
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PMID:Positive and negative regulation of gene expression in eukaryotic cells with an inducible transcriptional regulator. 927 20

The copZ gene of Bacillus subtilis encodes a copper chaperon CopZ that donates copper to the copper transporter CopA. Both genes copZ and copA are clustered to an operon and its promoter is regulated by Cu ions and CueR, a Mer-like transcriptional activator. Here we show that cadmium ions activate copZA expression as strong as copper ions. Northern hybridization analysis showed that copper and cadmium both induce the synthesis of a 2.7 kb copZA transcript and a 250 bp copZ transcript. A copA deletion mutant was sensitive to copper, whereas a copZ deletion resulted in an increased sensitivity to cadmium and copper. Transcription of the cadmium resistance gene cadA, which is adjacent to the copZA cluster, is extremely reduced in a copZ deletion strain. Transformation of copZ in trans restores wild type resistance to cadmium and copper in a copZ deletion strain. This excludes any polar effect and proves that the copZ encoded protein is important for copper and cadmium resistance.
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PMID:Metalloregulation in Bacillus subtilis: the copZ chromosomal gene is involved in cadmium resistance. 1521

The in-situ conformations of peptide layers formed from the adsorption of two different synthetic 15-mer peptides at the hydrophilic silicon oxide/aqueous solution interface have been determined using neutron reflectivity (NR). The first peptide is based on the native sequence of a protein-binding domain within a heteromeric transcriptional activator, HAP2, identified from yeast Saccharomyces cerevisiae, with tyrosine (Y) present at the 1st, 8th and 15th amino acid positions, hence we denote this YYY15. Substitution of tryptophan (W) at the same locations gives WWW15. Both peptides have alpha-helical structure in phosphate buffer, as determined by circular dichroism (CD) spectra. D(2)O was used as solvent in the NR experiments to highlight structural heterogeneity across the hydrogenated peptide layers. At pH 7, YYY15 was found to form a weakly adsorbed interfacial monolayer. However, the mutant WWW15 showed strong interfacial adsorption, with the interfacial layer characterized by a middle hydrophobic sublayer of 7-8 A with lower scattering length density and two almost symmetrical hydrophilic outer sublayers of 6-8 A with higher scattering length density, suggesting the formation of a "sideways-on" helical conformation. An increase in pH to 9 resulted in the improved packing within the interfacial layer with similar structure. However, decrease in pH to 5 reduced the interfacial adsorption, mainly due to the enhanced solubility of the peptides associated with the protonation of arginine (R) and lysine (K) groups and the decreasing concentration of divalent HPO(4)(2-) in the phosphate buffer. Subsequent assessment of the reversibility of adsorption showed that once the peptide layers were formed they did not desorb. These interfacial structures may provide feasible routes to interfacial nano-templating.
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PMID:Interfacial nano-structuring of designed peptides regulated by solution pH. 1526 24

We have investigated anaerobic respiration of the archaeal model organism Halobacterium sp. strain NRC-1 by using phenotypic and genetic analysis, bioinformatics, and transcriptome analysis. NRC-1 was found to grow on either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as the sole terminal electron acceptor, with a doubling time of 1 day. An operon, dmsREABCD, encoding a putative regulatory protein, DmsR, a molybdopterin oxidoreductase of the DMSO reductase family (DmsEABC), and a molecular chaperone (DmsD) was identified by bioinformatics and confirmed as a transcriptional unit by reverse transcriptase PCR analysis. dmsR, dmsA, and dmsD in-frame deletion mutants were individually constructed. Phenotypic analysis demonstrated that dmsR, dmsA, and dmsD are required for anaerobic respiration on DMSO and TMAO. The requirement for dmsR, whose predicted product contains a DNA-binding domain similar to that of the Bat family of activators (COG3413), indicated that it functions as an activator. A cysteine-rich domain was found in the dmsR gene, which may be involved in oxygen sensing. Microarray analysis using a whole-genome 60-mer oligonucleotide array showed that the dms operon is induced during anaerobic respiration. Comparison of dmsR+ and DeltadmsR strains by use of microarrays showed that the induction of the dmsEABCD operon is dependent on a functional dmsR gene, consistent with its action as a transcriptional activator. Our results clearly establish the genes required for anaerobic respiration using DMSO and TMAO in an archaeon for the first time.
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PMID:Genomic analysis of anaerobic respiration in the archaeon Halobacterium sp. strain NRC-1: dimethyl sulfoxide and trimethylamine N-oxide as terminal electron acceptors. 1571 36

Sugar signalling cascades are important components of regulatory networks in cells. Compared with the situation in bacteria, yeast and animals, participants of the sugar signalling pathways in plants are poorly understood. Several genes involved in starch synthesis are known to be sugar inducible, although the signal transduction pathways remain undisclosed. We reported recently the isolation of SUSIBA2, a transcription factor involved in sugar-mediated regulation of starch synthesis. Here, we used antisense oligodeoxynucleotide (ODN) inhibition, a powerful approach in medical sciences, to block the effects of SUSIBA2 in sugar-treated barley leaves. The uptake and intracellular trafficking of an 18-mer susiba2 antisense ODN in leaves were followed by confocal microscopy. Administration of the antisense ODN to the leaves impeded susiba2 expression by RNase H activation. This dramatically diminished the ectopic expression of the iso1 and sbeIIb genes and resulted in altered starch synthesis. This study illustrates the successful exploitation of the antisense ODN technology in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and identifies SUSIBA2 as a transcriptional activator in plant sugar signalling. Based on our findings, we propose a model for sugar-signalling control of starch synthesis.
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PMID:Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling. 1616 1

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