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PubMed2005

Crystal structures of the tryptophan repressor binding protein WrbA and complexes with flavin mononucleotide.

The tryptophan repressor binding protein WrbA binds to the tryptophan repressor protein TrpR. Although the biological role of WrbA remains unclear, it has been proposed to function in enhancing the stability of TrpR-DNA complexes. Sequence database analysis has identified WrbA as a founding member of a flavodoxin-like family of proteins. Here we present crystal structures of WrbA from Deinococcus radiodurans and Pseudomonas aeruginosa and their complexes with flavin mononucleotide. The protomer structure is similar to that of previously determined long-chain flavodoxins; however, each contains a conserved inserted region unique to the WrbA family. Interestingly, each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule.

Amino Acid Sequence↗

PubMed1997

Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor.

Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. We report the isolation, characterization and mapping of a human gene and its mouse homolog encoding a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, we demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. We demonstrate that repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia.

Amino Acid Sequence↗

PubMed1998

Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1.

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p.DNA. Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p.DNA.Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.

Base Sequence↗

PubMed1984

Nucleotide sequence of the gene, protein purification and characterization of the pSC101-encoded tetracycline resistance-gene-repressor.

The nucleotide sequence of the pSC101-encoded tetracycline repressor gene (tetR) was confirmed. The deduced amino acid sequence is compared to that of other repressor proteins. To overproduce the repressor protein, tetR was placed under the control of bacteriophage lambda promoter pL. Tet repressor protein was purified to homogeneity and shown to bind specifically to two tet operators and also to tetracycline (Tc). The inducer function of Tc is demonstrated by the loss of the specific binding between the tet operator DNA and the Tet repressor-Tc complex.

Amino Acid Sequence↗

PubMed1997

A model for extradenticle function as a switch that changes HOX proteins from repressors to activators.

The Drosophila EXD protein and its mammalian counterparts, the PBX proteins, have been proposed to function in HOX target selectivity. Here we show that exd function is required for the autoactivation phase of Dfd expression in the posterior head. Mutations that change the affinity of a small autoactivation element for EXD protein result in corresponding changes in the element's embryonic activity. Our data suggest that the EXD and DFD proteins directly activate this element in maxillary cells without cooperatively binding to a specialized heterodimer binding site. Based on the types of homeotic transformations and changes in gene expression observed in exd mutant embryos, we propose a new model for EXD/PBX action in which these proteins are required for HOX protein transcriptional activation functions, but dispensable for HOX transcriptional repression functions. Although the selection of a specific target gene by a HOX protein versus another may be explained in some cases by the selective modulation of HOX binding specificity by EXD, we favor the idea that EXD interacts in a more general sense with most HOX proteins to switch them into a state where they are capable of transcriptional activation.

Animals↗

PubMed1987

T lymphocyte response to bacteriophage lambda repressor cI protein. Recognition of the same peptide presented by Ia molecules of different haplotypes.

The murine T cell response to bacteriophage lambda cI repressor protein has been investigated. Isolation and characterization of class II-restricted T cell hybridomas from BALB/c and A/J mice undergoing a primary response has revealed that a single region of the protein, residues 12-26, is the immunodominant site. Fine specificity analysis using truncated peptides (P12-24 and P15-26) reveals a great deal of heterogeneity at the clonal level of I-Ad-restricted T cells. I-Ek-restricted T cells are less heterogeneous in their reactivity toward P12-24 and P15-26, but show diversity in their responses to peptide analogues with substitution at Tyr22. The specificity difference between T cell hybridomas of I-Ad-restriction and I-Ek-restriction and the inhibition effect of different inactive peptides suggest that the same peptide is presented in different configurations by different Ia molecules. Further, no cross-reactivity can be detected between T cells of these two haplotypes, Ia molecules and Ia bound-peptides.

Amino Acid Sequence↗

PubMed1998

Induction of CYP102 (cytochrome P450BM-3) in Bacillus megaterium by 17 beta-estradiol and 4-sec-butylphenol.

CYP102 (Cytochrome P450BM-3) is induced in Bacillus megaterium by barbiturates, peroxisome proliferators, and nonsteroidal anti-inflammatory drugs. We now describe the induction of CYP102 in B. megaterium by 17 beta-estradiol and by 4-sec-butylphenol. These estrogens interact with the repressor protein Bm3R1, causing it to dissociate with the operator of the CYP102 gene and allowing transcription to occur. We have developed a stable transfection of a construct into B. megaterium of a truncated CYP102 gene coupled with the luciferase gene in a promoterless plasmid and have used this construct to test the induction of CYP102 by these estrogens. Estradiol demonstrated a dose-dependent induction of CYP102 which saturated at a 2-fold increase at 150 microM 4 hr post-addition. 4-sec-Butylphenol produced a dose-dependent and time-dependent induction up to 300 microM and 6 hr post-induction.

Bacillus megaterium↗

PubMed2005

The Leukemia-associated ETO homologues are differently expressed during hematopoietic differentiation.

The Eight twenty-one (ETO) homologues are nuclear repressor proteins including ETO, myeloid-transforming gene-related protein 1 (MTGR1), and myeloid-transforming gene chromosome 16 (MTG16). ETO and MTG16 are both part of fusion proteins resulting from chromosomal translocations associated with acute myeloid leukemia. Expression of these chimeras results in a differentiation block that contributes to the onset of leukemia. In order to elucidate the relation between the ETO homologues and hematopoietic differentiation, we determined the expression of the homologues during differentiation of leukemic and normal hematopoietic cells. Our results showed MTGR1 and MTG16 to be ubiquitously expressed in leukemic cell lines, whereas expression of ETO was observed only in an erythroleukemic cell line. The MTGR1 and MTG16 proteins decreased during all trans-retinoic acid-, but not vitamin D(3)-induced differentiation of leukemic cells. The reduction seemed to reflect a decrease in transcript levels as well as in protein stability. MTGR1 transcripts were ubiquitously expressed in human bone marrow cells. The MTG16 transcripts of CD34(+) progenitor cells were rapidly downregulated by cytokine-induced differentiation into myeloid or erythroid lineages. ETO transcripts, present at very low abundance in CD34(+) progenitor cells, were transiently upregulated during erythroid differentiation. In conclusion, the differential expression of the ETO homologues suggests that they may have a potential role in hematopoietic differentiation.

Bone Marrow Cells↗

PubMed2004

Analysis of orthologous hrcA genes in Escherichia coli and Bacillus subtilis.

The hrcA gene codes for a transcriptional repressor protein interacting with the CIRCE operator thereby reducing expression of the groE operon of more than 120 bacterial species. At least in Bacillus subtilis, the activity of the HrcA protein is modulated by the GroE chaperonin system. We amplified the hrcA gene from five different bacterial species and analyzed its activity in Escherichia coli and Bacillus subtilis. While those from Clostridium acetobutylicum and Staphylococcus aureus turned out to be active, those of Helicobacter pylori, Lactococcus lactis and Thermotoga maritima were inactive in E. coli, but that of T. maritima turned out to repress expression of the reporter gene in B. subtilis. All these results strongly suggest to us a specific recognition of HrcA by the GroE chaperonin system.

Amino Acid Sequence↗

PubMed2003

Characterization and recombinant expression of the translational repressor RepB of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase in Comamonas testosteroni.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The gene of 3alpha-HSD/CR (hsdA) was cloned and characterized by our group. We have also reported that two repressor proteins (RepA and RepB) have been identified which regulate hsdA expression. To further characterize RepB, the protein was expressed in Escherichia coli and purified in an active state. Gel shift experiments showed that RepB binds to a 16 nucleotide sequence downstream of AUG of the hsdA mRNA, providing evidence that RepB acts on the translational level. The addition of testosterone to the culture medium led to a derepression. Furthermore, a plasmid was prepared containing a point mutation that inactivates only repA, but has no effect on hsdA, with which it happens to partly overlap. The result of coexpression experiments with this construct and a plasmid containing the genetic information for RepB showed that RepB is still active and is therefore not dependent on a functional RepA. In conclusion, RepB is a novel regulatory protein that inhibits the translation of hsdA mRNA, thereby leading to a decreased expression of 3alpha-HSD/CR.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

A proteinaceous gene regulatory thermometer in Salmonella.

Novel utilization of the coiled-coil motif is presented that enables TlpA, an autoregulatory repressor protein in Salmonella, to sense temperature shifts directly and thereby to modulate the extent of transcription repression. Salmonella cells shifted to higher temperatures, such as those encountered at host entry, showed derepressed tlpA activity. tlpA::lacZ fusions indicated that the promoter itself is insensitive to thermal shifts and that transcription control was exerted by the autorepressor TlpA only. In vitro studies with highly purified TlpA showed concentration and temperature dependence for both fully folded conformation and function, indicating that the thermosensing in TlpA is based on monomer-to-coiled-coil equilibrium.

Bacterial Proteins↗

PubMed1997

CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro.

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (>> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.

Alternative Splicing↗

PubMed2003

The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1.

In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H. jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.

Amino Acid Sequence↗

PubMed1990

31P NMR spectra of an oligodeoxyribonucleotide duplex lac operator-repressor headpiece complex.

The interaction of a symmetric lac operator duplex, d(TGTGAGCGCTCACA)2, with the N-terminal 56-residue headpiece fragment of the lac repressor protein was monitored by 31P NMR spectroscopy. The changes in the 31P chemical shifts upon addition of the headpiece demonstrated an end point of two headpiece fragments per symmetric 14-mer duplex with each headpiece binding to the T1pG2pT3pG4pA5 ends of the duplex. The specific phosphate 31P perturbations observed are consistent with those residues implicated in protein binding by previous NMR, molecular biological, and biochemical techniques. Upon complexation, the 31P signals of phosphates G2-A5 showed upfield or downfield shifts (less than 0.2 ppm) while most other residues were unperturbed. The interactions were dependent on ionic strength. The 31P NMR data provide direct evidence for predominant recognition of the 5' strand of the 5'-TGTGA/3'-ACACT binding site.

Bacteriophage lambda↗

PubMed2001

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.

Allosteric Regulation↗

PubMed1997

Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants.

This study investigated whether an increased production of the plant hormone cytokinin in roots, the main site of its synthesis and putative signaling organ, can influence developmental events, such as growth of axillary shoot meristems or leaf senescence, in the plant shoot. To this end, transgenic tobacco plants (Nicotiana tabacum L.) were generated that conditionally overproduce cytokinins. These plants harbour the ipt gene under the transcriptional control of a modified 35S promoter that is repressed in plants with high titers of tetracycline repressor protein. De-repression of transcription led to a rapid more than 50-fold increase of hormone concentration. The time course of changes in the steady-state levels of 16 different cytokinin metabolites, as a consequence of IPT enzyme activity, was monitored in different plant tissues. Zeatin riboside was the first and most dramatically increased product; zeatin, dihydrozeatin and glucosides accumulated later. The consequences of enhanced cytokinin synthesis remained mainly restricted to the site of hormone production. For example, de-repression of ipt gene transcription in lateral buds caused the growth of single buds only at the site of tetracycline application. In reciprocal grafts of transgenic plants with wild-type plants, no biological cytokinin effects, i.e. growth of lateral shoot meristems or sequential leaf senescence, were observed in the non-transgenic plant part. Also, the increase in steady-state levels of cytokinins remained restricted mainly to the transgenic part, despite a specific increase of the zeatin riboside concentration in the transpiration stream. These results question the role of cytokinins as a long-range root-to-shoot signal in correlative control of apical dominance and sequential leaf senescence of tobacco, and support the assumption that this hormone is relevant to paracrine signaling.

Aging↗

PubMed2001

Brinker requires two corepressors for maximal and versatile repression in Dpp signalling.

decapentaplegic (dpp) encodes a Drosophila transforming growth factor-beta homologue that functions as a morphogen in the developing embryo and in adult appendage formation. In the wing imaginal disc, a Dpp gradient governs patterning along the anteroposterior axis by inducing regional expression of diverse genes in a concentration-dependent manner. Recent studies show that responses to graded Dpp activity also require an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Here we show that Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, we demonstrate that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. Our results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters.

Alcohol Oxidoreductases↗

PubMed1988

Repression of the aroF promoter by the TyrR repressor in Escherichia coli K-12: role of the 'upstream' operator site.

Three sequences are required for complete repression of the aroF promoter by the TyrR repressor protein. Two of these operator sites lie adjacent to each other and overlap the -35 region of the aroF promoter while the third lies about 70 base pairs upstream of the promoter. An aroF-cat (chloramphenicol acetyltransferase) gene fusion has been used to assay the effect of DNA insertions that alter the distance between the two promoter-proximal and the third, distal, operator sites on the repression of the aroF promoter in vivo. The distal site contributes to the repression of the promoter up to a distance of about 400 base pairs and its effect is not dependent on its separation from the first and second sites by an integral number of turns of the DNA helix.

Acetyltransferases↗