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DNA binding by the coliphage 186 repressor protein CI.

The cI gene of coliphage 186 maintains lysogeny and confers immunity to 186 infection by repressing the major early promoter, p(R), and the promoter for the late transcription activator gene, p(B). Gel mobility shirt and DNase I footprinting show that CI protein binds to the DNA at p(R) and p(B) and also to sites approximately 300 base pairs upstream and downstream of p(R), called FL and FR. Mutations which cause virulence reduce CI binding to p(R). The biochemical and genetic data identify three CI operators at p(R), two at p(B), and single operators at FL and FR. The operators at the p(B), FL, FR, and central p(R) sites are inverted repeat sequences, separated by 5 base pairs (Type A) or, in the case of p(R), by 4 base pairs (Type A'). A different inverted repeat operator sequence (Type B) is proposed for the binding sites on each side of the central site at p(R). Thus, CI appears to recognize two distinct DNA sequences. CI binds cooperatively to adjacent operators, and binding at p(R) is strongly dependent on these cooperative interactions. A high order CI multimer appears to be the active DNA binding species, even at single operators.

Amino Acid Sequence↗

Immobilisation of a repressor protein for binding of plasmid DNA.

The use of plasmid DNA in gene therapy and genetic vaccination has increased the need for scalable and sustainable production processes. One key challenge for bioprocess engineering is the separation of plasmid DNA from structurally related impurities. Affinity purification procedures allow a highly selective capturing of the target molecule. In this paper, we present the isolation of a his-tagged lac repressor, its non-covalent immobilisation to different matrices and binding of DNA, thus enabling us to screen for combinations of ligands and stationary phases by using a building block principle.

Affinity Labels↗

Expression, purification and characterization of the membrane-associated HrcA repressor protein of Helicobacter pylori.

Helicobacter pylori, a microaerophilic, gram-negative bacterium is a human pathogen that colonizes the gastric niche and is associated with several acute and chronic stomach diseases. In order to survive in the gastric environment and become pathogenic, the bacterium relies on a plethora of virulence factors, which also include heat shock proteins. We previously showed that two out of the three operons encoding the major cellular chaperone machineries are transcriptionally repressed by two regulators, HrcA and HspR. Till now, molecular studies aimed at the understanding of the role of each protein in controlling transcription was hampered by toxicity and insolubility of HrcA in heterologous expression systems. Similar problems were encountered by many other groups studying HrcA from different bacteria. In this study, we analyzed the amino acid sequence of HrcA that predicted association of this protein to the inner membrane, which was experimentally verified. Subsequently, we implemented a dedicated induction protocol which enabled the overexpression of the recombinant His-HrcA protein in the soluble fraction of Escherichia coli cells. Moreover, we developed a purification procedure for His-HrcA that allowed us to obtain highly pure preparation of the protein. The functionality of the purified protein was then confirmed with an in vitro DNA-binding assay.

Amino Acid Sequence↗

Presence of a repressor protein for testis-specific H2B (TH2B) histone gene in early stages of spermatogenesis.

The rat testis-specific TH2B histone gene assumes a hypomethylated chromatin structure at all stages of spermatogenesis. However, the TH2B mRNA level is very low in pre-meiotic spermatogenic cells and rises sharply in meiotic pachytene spermatocytes. The low level of TH2B mRNA in pre-meiotic spermatogenic cells appears to be a result of transcriptional repression of the gene by a pre-meiotic cell-specific protein which binds to a site between the TATA element and the transcription initiation site of TH2B gene.

Animals↗

An electron spin resonance study of the Mn(II) and Cu(II) complexes of the Fur repressor protein.

EPR spectra of Mn(II) Fur complex suggested the presence of Mn(II) in one site per Fur monomer in which Mn(II) is present in a low symmetry environment. The binding of the Mn(II) Fur complex to a DNA fragment "iron box" has a slight broadening effect on the Mn(II) signal and hence it altered the symmetry of the Mn(II) environment. We also report EPR spectra of Cu(II) Fur and Cu(II) C92S C95S mutant Fur complexes as models for Fe(II) complexes; the anisotropic g values and A values observed indicate the presence of Cu(II) in two different environments in the protein; a major axially distorted Cu(II) site bound to nitrogen and a minor distorted tetrahedral sulfur bound to the Cu(II) site. The effect of metal ion on Fur DNA binding is also discussed.

Base Sequence↗

Mutant Escherichia coli arginine repressor proteins that fail to bind L-arginine, yet retain the ability to bind their normal DNA-binding sites.

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.

Amino Acid Sequence↗

Transcriptional regulation of the c-jun gene by AP-1 repressor protein JDP2 during the differentiation of F9 cells.

ATF-2 and p300 cooperate in the activation of transcription of the c-jun gene during the differentiation of F9 cells. We show here that a repressor of AP-1, JDP2 (Jun dimerization protein 2), inhibits the transactivation of the c-jun gene by ATF-2 and p300, by recruitment of a histone deacetylase complex, thereby repressing the retinoic acid (RA)-induced transcription of the c-jun gene and inhibiting the RA-mediated differentiation of F9 cells. Furthermore, JDP2/HDAC complex was replaced by p300 complex on the DRE (differentiation response element) of the c-jun promoter within 24 h after the start of exposure of cells to RA, with an accompanying change in the histone-acetylation status of the chromatin, indicating that the initiation of transcription of the c-jun gene was controled by sequential action of HDAC(s) and HAT(s) on c-jun promoter.

Animals↗

Purification and characterization of the major 50-kDa repressor protein from cytoplasmic mRNP of rabbit reticulocytes.

A 50-kDa protein has been purified to homogeneity from free mRNP of rabbit reticulocytes. This protein, designated as p50, is present within both free mRNP (approximately 4 mol protein/mol globin mRNA) and polyribosomal mRNP (approximately 2 mol protein/mol globin mRNA). p50 is a basic protein (pI approximately 9.5) and is characterized by a high glycine content of approximately 20%. Nitrocellulose-filter analysis has shown that p50 interacts with globin mRNA with an association constant of approximately 2.5 x 10(8) M-1 (100 mM KAc, 4 degrees C). Various RNA and polyribonucleotides have the following relative affinity for p50; poly(G) > poly(U) > globin mRNA approximately 16S rRNA > poly(A) > poly(C). p50 can be phosphorylated both in vitro and in vivo.

Amino Acids↗

An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.

Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.

Amino Acid Sequence↗

Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells.

Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process.

Breast Neoplasms↗

Reconstructing repressor protein levels from expression of gene targets in Escherichia coli.

The basic underlying problem in reverse engineering of gene regulatory networks from gene expression data is that the expression of a gene encoding the regulator provides only limited information about its protein activity. The proteins, which result from translation, are subject to stringent posttranscriptional control and modification. Often, it is only the modified version of the protein that is capable of activating or repressing its regulatory targets. At present there exists no reliable high-throughput technology to measure the protein activity levels in real-time, and therefore they are, so-to-say, lost in translation. However, these activity levels can be recovered by studying the gene expression of their targets. Here, we describe a computational approach to predict temporal regulator activity levels from the gene expression of its transcriptional targets in a network motif with one regulator and many targets. We consider an example of an SOS repair system, and computationally infer the regulator activity of its master repressor, LexA. The reconstructed activity profile of LexA exhibits a behavior that is similar to the experimentally measured profile of this repressor: after UV irradiation, the amount of LexA substantially decreases within a few minutes, followed by a recovery to its normal level. Our approach can easily be applied to known single-input motifs in other organisms.

Bacterial Proteins↗

Dissecting the molecular details of prokaryotic transcriptional control by surface plasmon resonance: the methionine and arginine repressor proteins.

The commercial surface plasmon resonance (SPR) biosensors, BIACORE, have been used to investigate the molecular details of macromolecular interactions at prokaryotic promoter-operators. For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of the protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexes being formed. The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-operator fragment; with transcripts collected and subsequently characterised by RT-PCR. This technique has enabled us to investigate how repressor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcription. Remarkably, the repression complex appears to stabilise binding of RNAP, whilst having the expected effects on the levels of transcripts produced. This may well be a general mechanism allowing rapid transcription initiation to occur as soon as the repression complex dissociates. These techniques have also been used to examine protein-DNA interactions in the E. coli and Bacillus subtilis arginine repressor systems. The repressors are the products of the argR and ahrC genes, respectively. Both proteins form hexamers in rapid equilibrium with smaller subunits believed to be trimers. There are three types of operator in these systems, autoregulatory, biosynthetic and catabolic (B. subtilis only). Sensorgrams show that each protein recognises the three types of immobilised operator differently and that binding is stimulated over 100-fold by the presence of L-arginine.

Bacterial Proteins↗

The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori.

In the present study, we provide evidence that the groESL, hrcA-grpE-dnaK and cbpA-hspR-orf operons encoding the major chaperones of the human gastric pathogen Helicobacter pylori are transcribed by the vegetative sigma factor sigma80 and are regulated negatively by the transcriptional repressor HspR. In vitro studies with purified recombinant HspR protein established that the protein represses transcription by binding to large DNA regions centred around the transcription initiation site in the case of the Pcbp promoter, and around -85 and -120 in the case of the Pgro and Phrc promoters respectively. All three binding sites contain DNA motifs with some similarity to the HAIR sequence identified as a consensus for the HspR protein of Streptomyces. In contrast to the situation in Streptomyces, in which transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible by thermal stimuli. Transcription of the groESL and cbpA-hspR-orf operons is induced by osmotic shock, while transcription of the hrcA-grpE-dnaK operon, although HspR dependent, is not affected by salt treatment. The possibility that HspR could constitute a global transcriptional regulator for diverse cellular functions with implications for pathogenesis is discussed.

Bacterial Proteins↗

The Saccharomyces cerevisiae DAL80 repressor protein binds to multiple copies of GATAA-containing sequences (URSGATA).

Induced expression of the allantoin (DAL) catabolic genes in Saccharomyces cerevisiae has been suggested to be mediated by interaction of three different types of promoter elements. First is an inducer-independent upstream activation sequence, UASNTR, whose operation is sensitive to nitrogen catabolite repression. The GLN3 product is required for UASNTR-mediated transcriptional activation. This site consists of two separated elements, each of which has a GATAA sequence at its core. Response of the DAL genes to inducer is mediated by a second type of cis-acting element, DAL UIS. The DAL82 and DAL81 genes are required for response to inducer; DAL82 protein is the UIS-binding protein. When only the UASNTR and UIS elements are present, DAL gene expression occurs at high levels in the absence of inducer. We, therefore, hypothesized that a third element, an upstream repressor sequence (URS) mediates maintenance of DAL gene expression at a low level when inducer is absent. Since the DAL and UGA genes are overexpressed and largely inducer independent in dal80 deletion mutants, we have suggested DAL80 protein negatively regulates a wide spectrum of nitrogen-catabolic gene expression, likely in conjunction with a URS element. Here we show that DAL80 protein binds to DAL3 and UGA4 upstream DNA sequences, designated URSGATA, consisting of two GATAA-containing sites separated by at least 15 bp. The preferred orientation of the sites is tail to tail, but reasonable binding activity is also observed with a head-to-tail configuration. URSGATA elements contain the sequence GATAA at their core and hence share sequence homology with UASNTR elements.

Amino Acid Sequence↗

m-Bop, a repressor protein essential for cardiogenesis, interacts with skNAC, a heart- and muscle-specific transcription factor.

The m-Bop protein encoded by the mouse Bop gene is strongly expressed in heart and skeletal muscle, and recent studies with Bop knockout mice have demonstrated that m-Bop is essential for cardiogenesis in vivo and can act as a HDAC-dependent repressor in vitro. In the present studies, m-Bop was observed to interact with skNAC, a reported transcriptional activator specific to heart and skeletal muscle. The amino-terminal S region of the split S-ET domain of m-Bop as well as the MYND domain were required for interaction with skNAC in both the two-hybrid system and in coimmunoprecipitation experiments from cultured mammalian cells. As shown previously for interaction of the MYND domain-containing transcriptional corepressor, BS69, with several viral and cellular oncoproteins, a PXLXP motif in skNAC was required for interaction with m-Bop. Similar kinetics of induction and localization of m-Bop and skNAC during the induction of myogenesis in cultured C2C12 cells suggests a possible associated role for these proteins during this process.

Animals↗

Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation.

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.

Animals↗

Stat3 enhances vimentin gene expression by binding to the antisilencer element and interacting with the repressor protein, ZBP-89.

Vimentin exhibits a complex pattern of developmental- and tissue-specific expression and is aberrantly expressed in most metastatic tumors. The human vimentin promoter contains multiple DNA elements, some of which enhance gene expression and one that inhibits. A silencer element (at -319) binds the repressor ZBP-89. Further upstream (at -757) is an element, which acts positively in the presence of the silencer element and, thus, is referred to as an antisilencer (ASE). Previously, we showed that Stat1alpha binds to this element upon induction by IFN-gamma. However, substantial binding and reporter gene activity was still present in nontreated cells. Here, we have found that Stat3 binds to the ASE element in vitro. Transfection experiments in COS-1 cells with various vimentin promoter--reporter constructs show that gene activity is dependent upon the cotransfection and activation of Stat3. Moreover, activated Stat3 can overcome ZBP-89 repression. Coimmunoprecipitation studies demonstrate that Stat3 and ZBP-89 can interact and confocal microscopy detects these factors to be colocalized in the nucleus. Moreover, a correlation exists between the presence of activated Stat3 and vimentin expression in MDA-MB-231 cells, which is lacking in MCF7 cells where vimentin is not expressed. In the light of these results, we propose that the interaction of Stat3 and ZBP-89 may be crucial for overcoming the effects of the repressor ZBP-89, which suggests a novel mode for Stat3 gene activation.

Animals↗

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum.

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EM-SAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903-10907]. All cell-free extracts formed high-molecular weight protein-DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein-DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA-protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a "mycoparasitic" protein-protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein-DNA complex.

Base Sequence↗