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PubMed1998

A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2).

whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.

Amino Acid Sequence↗

PubMed2004

LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

Bacterial Proteins↗

PubMed1987

Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the arginine repressor.

In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis. We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated. The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ("ARG boxes"). The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined. Antibodies prepared against the fusion protein react with the repressor. The repressor is precipitable by L-arginine, which facilitates its purification. The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500. To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR.

Amino Acid Sequence↗

PubMed1982

Purification and properties of the biotin repressor. A bifunctional protein.

Definitive evidence is presented for the bifunctional nature of the biotin repressor protein which possesses both regulatory and enzymatic activities. The repressor protein can activate biotin in the presence of ATP to form biotinyl-5'-adenylate, the co-repressor which remains tightly bound to the repressor protein. This complex can either bind to the operator site and inhibit transcription or transfer the biotinyl moiety to a lysine residue of the apoenzyme of acetyl-CoA carboxylase. The two activities were coincident throughout a purification procedure which resulted in a 3500-fold increase in activity. Gel electrophoresis of the purified preparation, under native or denaturing conditions, showed three proteins with the activity corresponding to the major protein band of apparent Mr = 34,000. On gel exclusion chromatography, the activity was also associated with a protein of Mr varying fro 37,000-44,000, indicating the protein is monomeric. The occasional appearance of multiple bands with biological activity in the native gels suggests that the repressor protein can also exist in multimeric forms. On chromatofocusing, the repressor activity and the holoenzyme synthetase activity were coincidental, with the peak of activity at pH 7.2, the isoelectric point. Only a single protein band with Mr = 34,000 was observed on SDS gel electrophoresis of all fractions showing activity.

Acetyl-CoA Carboxylase↗

PubMed1984

Trp holorepressor-trp operator interaction studied by protein distribution analysis.

Trp repressor protein of Escherichia coli (Mr 24,700) undergoes a conformational change upon interaction with L-tryptophan that enables the resulting binary complex to bind with high specificity to several operator targets in double-stranded DNA. By protein distribution analysis it was shown that a significant fraction of Trp repressor is inert in operator binding. The equilibrium dissociation constant for Trp holorepressor-Trp operator interaction is 6.7 nM at 20 degrees in 0.05M NaCl, pH 7.4. The Trp holorepressor-trp operator complex consists of one molecule of each of the participating species, even at high molar ratios of protein to DNA.

Bacterial Proteins↗

PubMed1991

The RepA repressor can act as a transcriptional activator by inducing DNA bends.

We have shown that a transcriptional repressor protein can regulate promoter activity via DNA bending by using the pLS1 plasmid promoter PII (which has intrinsic curvature upstream of its -35 box) and the plasmid-encoded repressor protein RepA (which strongly bends DNA). Substitution of the curved region for a straight DNA fragment containing the RepA target resulted in increased (or decreased) gene expression when RepA was supplied in trans: enhanced gene expression was evident when the target of RepA and the promoter were on the same face of the DNA helix; repression was found when they were on opposite faces of the DNA. In vitro activation of transcription from PII was observed when supercoiled DNA was used as template, but not with linear molecules. We propose that promoter activity can be regulated by the proper positioning (in or out of phase) of an induced DNA bend with the RNA polymerase recognition sites.

Bacterial Proteins↗

PubMed2001

A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression 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 enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli. Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3alpha-HSD/CR in C. testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.

3-Hydroxysteroid Dehydrogenases↗

PubMed1979

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.

Amino Acids↗

PubMed2001

Regulation of apoA1 gene expression with acidosis: requirement for a transcriptional repressor.

Serum apolipoprotein A(1) (apoA(1)) concentration is inversely correlated with the risk of premature atherosclerosis. Serum apoA(1) concentrations are regulated, in part, at the transcriptional level. ApoA(1) mRNA is synthesized primarily in the liver and small intestine, under the direction of a number of signaling molecules and tissue-specific regulatory elements. Previously, we demonstrated that extracellular acidosis suppresses apoA(1) mRNA levels at the level of transcription. Here we demonstrate that intracellular acidosis, in the absence of extracellular pH changes, represses apoA(1) promoter activity. Repression occurs through a pH responsive element (pH-RE) located within the apoA(1) gene promoter. Acidosis increases the specific DNA binding activity of a putative repressor protein within the immediate 5'-flanking region of the apoA(1) gene. The cis-element that binds the putative repressor protein contains a negative thyroid hormone response element (nTRE) located 3' and adjacent to the apoA(1) TATA box. Mutation of the nTRE/pH-RE abrogates protein binding and alters the activity of reporter genes controlled by this element. Repression by acidosis did not require de novo mRNA and protein synthesis. Inhibition of tyrosine kinase activity and diacylglycerol-stimulated protein kinase C (PKC) signaling pathways with tyrophostin A47 and phorbol myristate acetate, respectively, did not affect the repression of apoA(1) promoter activity with acidosis. These results suggest that transcriptional repression of the apoA(1) gene by alterations in ambient pH is associated with enhanced DNA binding activity of a repressor protein, through a mechanism which appears to be independent of de novo mRNA and protein synthesis, tyrosine kinase activity, or PKC activation.

Acidosis↗

PubMed2002

Expression of a 434:VP16 chimeric activator leads to high-level activation of gene expression in stable transformants of Arabidopsis.

The performance of an expression system based on a fusion of the bacteriophage 434-repressor to the VP16 activation domain of Herpes simplex virus type 1 (434:VP16) was tested after stable integration into Arabidopsis. A special feature of this system was the use of the monocot maize ubiquitin1 and rice actin1 promoters to drive the expression of the 434:VP16 activator and 434-repressor, respectively. Our results demonstrate that the maize ubiquitin1 and the rice actin1 promoters, each of which contain introns, are active in Arabidopsis and can be used to express genes in this dicot species. Activation of gene expression after co-integration of the activator and reporter cassettes into the same genomic locus resulted in a higher activation level (84-fold activation) compared to crossing individual lines expressing only the activator or the operator reporter cassette alone (9-fold activation). Increasing the number of operator elements in the reporter cassette from 1 to 4 increased the activation level in cross-activated lines to an average of 281-fold with one combination of parental lines giving a 900-fold activation. Simultaneous expression of the 434-repressor protein driven by the rice actin promoter resulted in a significant decrease in the 434:VP16 mediated reporter gene expression. Nevertheless, an overall induction via 434:VP16 was possible even in the presence of the 434-repressor protein. This feature is important for genes which need to be absolutely repressed except under activating conditions. To our knowledge this investigation is the first report on the use of the 434:VP16 chimeric activator in an expression system in stably transformed plant lines.

Arabidopsis↗

PubMed1984

Demonstration of two operator elements in gal: in vitro repressor binding studies.

Genetic and DNA base sequence analyses of cis-dominant mutations that derepress the gal operon of Escherichia coli suggested the existence of two operator loci needed for gal repression. One (OE) is located immediately upstream to the two overlapping gal promoters and the other (OI) is inside the first structural gene. We have investigated the ability of wild-type and mutant OE and OI DNA sequences to bind to gal repressor. The repressor has been purified from cells containing a multicopy plasmid in which the repressor gene is brought under the control of phage lambda PL promoter. The DNA-repressor interactions are detected by the change in electrophoretic mobility of labeled DNA that accompanies its complex formation with repressor protein. The purified repressor shows concentration-dependent binding to both O+E and O+I but not to OEc and OIc sequences. These results authenticate the proposed operator role of the two homologous gal DNA control elements and thereby establish that the negative control of the gal operon requires repressor binding at both OE and OI, which are separated by greater than 90 base pairs.

Electrophoresis, Polyacrylamide Gel↗

PubMed1990

Affinity purification of specific DNA fragments using a lac repressor fusion protein.

A new method for purification of specific DNA sequences using a solid phase technique has been developed based on a fusion between the Escherichia coli lac repressor gene (lacI) and the staphylococcal protein A gene (spa). The fusion protein, expressed in Escherichia coli, is active both in vivo and in vitro with respect to its three functional activities (DNA binding, IPTG induction, and IgG binding). The recombinant protein can be immobilized in a one-step procedure with high yield and purity using the specific interaction between protein A and the Fc-part of immunoglobulin G. The immobilized repressor can thereafter be used for affinity purification of specific DNA fragments containing the lac operator (lacO) sequence.

Base Sequence↗

PubMed1993

The pathogenesis of superficial siderosis of the central nervous system.

In advanced cases of superficial siderosis of the human central nervous system, the clinical triad of hearing loss, cerebellar ataxia, and myelopathy permits the diagnosis at the bedside, and magnetic resonance imaging readily confirms the hemosiderin deposits in brainstem, cerebellum, and spinal cord. To study the pathogenesis of this condition and explain the selective vulnerability of the cerebellum, experimental siderosis was induced in rabbits by the repeated intracisternal injection of autologous red blood cells. The earliest cellular response in the cerebellar molecular layer was hyperplasia and hypertrophy of microglia as displayed by immunocytochemistry for ferritin. Microglia also contained iron, but ferritin biosynthesis appeared to proceed without commensurate iron accumulation. This early apoferritin response probably occurred due to the presence of heme, rather than iron, in the cerebrospinal fluid and subpial tissue. Ferritin biosynthesis is accelerated when the ferritin repressor protein is dissociated from ferritin messenger ribonucleic acid. A specific antiserum localized ferritin repressor protein predominantly to astrocytes including Bergmann glia. It is proposed that abundance and proximity of ferritin repressor protein--immunoreactive Bergmann glia and ferritin-containing microglia in the cerebellar molecular layer permit prompt cellular interaction in the conversion of heme to ferritin and ultimately hemosiderin.

Animals↗

PubMed1998

Functional characterization of the human CYP1A1 negative regulatory element: modulation of Ah receptor mediated transcriptional activity.

The mechanisms that underly the regulation of human CYP1A1 have merited considerable attention because of their association both with toxic outcomes and the etiology of several cancers. Previous work conducted in this laboratory has identified a negative regulatory element (NRE) in the 5' region of this gene that appeared to modulate CYP1A1 transcriptional activity. This NRE is present in two functional copies, a high affinity 21-bp palindrome centered at position -784, and an additional element found within a GC-rich region between position -728 and -558. In this report, the regulatory function of the NREs in the context of the CYP1A1 promoter was evaluated. This was accomplished by substituting mutated elements for the corresponding wild-type element in a vector that contained human CYP1A1 sequences positions -1140 to +59 directing the transcription of the chloramphenicol acetyltransferase reporter gene. Expression vectors containing specific mutations in each or both NREs were characterized. We show that eliminating the binding of the CYP1A1 repressor protein to one or both repressor motifs results in a significant 2- to 3-fold increase in the inducibility of CYP1A1 promoter activity. Although mutation of both sites appeared to result in an increase in inducibility over that observed with only one site mutated, the effect was not additive. Such aberrant transcriptional activity correlates with the highly inducible aryl hydrocarbon hydroxylase phenotype that is a reported marker for individuals predisposed to lung cancer. Mutation of the NRE, or more likely, the cognate repressor protein(s), may provide a genetic basis for this phenotype.

Base Sequence↗

PubMed1992

Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli.

The DNA sequence encoding the rbs repressor protein, RbsR, has been determined. Amino acid sequence analyses of the product of an rbsR-lacZ fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein. DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon. RbsR is a member of a family of homologous repressor proteins having very similar DNA-binding sites and binding to similar operator sequences.

Amino Acid Sequence↗

PubMed1997

Ligand-induced conformational changes in lactose repressor: a fluorescence study of single tryptophan mutants.

A key element in the ability of lac repressor protein to control transcription reversibly is the capacity to assume different conformations in response to ligand binding. To investigate regions of the protein involved in these conformational changes, mutant repressor proteins containing single tryptophans were created by mutating each of the two native tryptophan residues to tyrosine and changing the residue of interest to tryptophan. Tryptophans substituted in the following locations were highly accessible to quenchers with no changes in fluorescence or quenching properties in the presence of ligands: in the N-terminal helix-turn-helix for Y7, at the junction between the N-terminus and N-subdomain for L62, in the N-subdomain of the monomer-monomer interface for residue E100 or Q117, or at the C-terminal region for K325. Tryptophan at position F226 in the C-subdomain subunit interface was only moderately exposed to quenchers and unresponsive to ligands. In contrast, the fluorescence and quenching properties of single tryptophans placed in the central region of the protein were affected by ligands. Inducer binding altered the accessibility to quencher for tryptophan at H74 or F293, but no changes were detected upon binding operator. Exposure of tryptophan at the position occupied by Y273 was affected by both inducer and operator, indicating alterations in this region by both ligands. These results suggest that, in the areas of the lac repressor probed by these substitutions, the inducer-bound form differs from the conformation of the unliganded form.

Bacterial Proteins↗

PubMed1995

The solution structure of the Mu Ner protein reveals a helix-turn-helix DNA recognition motif.

BACKGROUND: The Mu Ner protein is a small (74 amino acids), basic, DNA-binding protein found in phage Mu. It belongs to a class of proteins, the cro and repressor proteins, that regulate the switch from the lysogenic to the lytic state of the phage life cycle. There is no significant sequence identity between Mu Ner and the cro proteins of other phages, despite their functional similarity. In addition, there is no significant sequence identity with any other DNA-binding proteins, with the exception of Ner from the related phage D108 and the Nlp protein of Escherichia coli. As the tertiary structures of Mu Ner and these two related proteins are unknown, it is clear that a three-dimensional (3D) structure of Mu Ner is essential in order to gain insight into its mode of DNA binding. RESULTS: The 3D solution structure of Mu Ner has been solved by 3D and 4D heteronuclear magnetic resonance spectroscopy. The structure consists of five alpha helices, two of which comprise a helix-turn-helix (HTH) motif. Analysis of line broadening and disappearance of crosspeaks in a 1H-15N correlation spectrum of the Mu Ner-DNA complex suggests that residues in these two helices are most likely to be in contact with the DNA. CONCLUSIONS: Like the functionally analogous cro proteins from phages lambda and 434, the Mu Ner protein possesses a HTH DNA recognition motif. The Ner protein from phage D108 and the Nlp protein from E. coli are likely to have very similar tertiary structures due to high amino-acid-sequence identity with Mu Ner.

Amino Acid Sequence↗

PubMed1986

Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.

A method has been developed for making "footprints" of proteins bound to DNA. The hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II), is the reagent used to cut the DNA. Hydroxyl radical breaks the backbone of DNA with almost no sequence dependence, so all backbone positions may be monitored for contact with protein. In addition to defining the DNA sequence in contact with the protein, hydroxyl radical footprints embody structural information about the DNA-protein complex. For example, hydroxyl radical footprints of the bacteriophage lambda repressor and Cro protein show directly that these proteins are bound to only one side of the DNA helix. Additional contacts of lambda repressor and Cro protein with DNA, not observed by other chemical footprinting methods, are revealed by hydroxyl radical footprinting.

Binding Sites↗