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PubMed1996

The mechanism of CAP-lac repressor binding cooperativity at the E. coli lactose promoter.

The cyclic AMP receptor protein (CAP) and lactose repressor bind their regulatory sites in the lactose promoter with moderate cooperativity (omega C101 = 11.8(+/- 3.7)). This cooperativity is significantly reduced by the removal of DNA located upstream of the CAP binding site or by substitution of the dimeric lacI-18 mutant repressor for the wild-type tetrameric protein. These results are consistent with a mechanism of interaction in which CAP bends the DNA and the lac repressor binds simultaneously to its operator site and to promoter-distal sequences. Similar values of omega C101 were obtained with a promoter truncation containing the O3 pseudooperator site and one in which the site is destroyed, suggesting that DNA contacts distal to the O3 site are necessary for cooperative binding.

Binding Sites↗
PubMed1995

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition.

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of beta-galactosidase with symmetric variants of alpha- and beta-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the alpha-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the alpha-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. A180, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for alpha-centered trp operator variants with exchanges in positions 3, 4 and 5.

Amino Acid Sequence↗
PubMed2003

An anti-repression Fur operator upstream of the promoter is required for iron-mediated transcriptional autoregulation in Helicobacter pylori.

The Fur protein acts as a regulator of iron-dependent gene transcription in bacteria. In Helicobacter pylori, Fur regulates iron-activated and iron-repressed promoters. It also acts as an autoregulatory rheostat of transcription to fine-tune its own expression in response to iron by binding to three operators at its own promoter Pfur. Using biochemical and genetic analyses, here we show that the distal upstream operator III (centred at -110) is essential for iron regulation of Pfur and functions as an anti-repression site that is bound by the iron-free form of Fur to induce transcription. Furthermore, operator I (centred at -50) may have a dual role both as a high-affinity binding site for Fur and as an UP element. We propose that its role is ensuring that Fur expression is not repressed below a minimum threshold level. Our data supports a novel promoter architecture and mechanism of regulation by Fur.

Bacterial Proteins↗
PubMed1989

Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.

Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained approximately 2 x 10(7) active repressor molecules (and 1-2 x 10(4) copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of beta-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the beta-galactosidase gene under control of the optimal lacO promoter was constructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable beta-galactosidase. Addition of isopropyl beta-D-thiogalactoside restored expression to greater than 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.

DNA, Recombinant↗
PubMed2006

Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction.

In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer-repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer-repressor stoichiometry in induction of the lac operon.

DNA, Bacterial↗
PubMed1994

Specificity determinants for the interaction of lambda repressor and P22 repressor dimers.

The related phage lambda and phage P22 repressors each bind cooperatively to adjacent and separated operator sites, an interaction that involves a pair of repressor dimers. The specificities of these interactions differ: Each dimer interacts with its own type but not with dimers of the heterologous repressor. The two repressors exhibit significant amino acid sequence homology in their carboxy-terminal domains, which are responsible for both dimer formation and the dimer-dimer interaction. Here, we identify a collection of amino acid substitutions that disrupt the protein-protein interaction of DNA-bound lambda repressor dimers and show that several of these substitutions have the same effect when introduced at the corresponding positions of P22 repressor. We use this information to construct a variant of the lambda repressor bearing only six non-wild-type amino acids that has a switched specificity; that is, it binds cooperatively with P22 repressor, but not with wild-type lambda repressor. These results identify a series of residues that determine the specificities of the two interactions.

Amino Acid Sequence↗
PubMed1988

Mutation in a locus linked to penB-nmp causes suppression of the Mtr phenotype of Neisseria gonorrhoeae.

The chromosomal locus mtr, which encodes low-level resistance to multiple antibacterial agents in Neisseria gonorrhoeae, is subject to phenotypic suppression by env mutations that increase the permeability of the envelope. We have identified a new locus, mom (for modifier of Mtr), which is located on the chromosome very close to penB and nmp, loci known to be linked to each other and to spc. Phenotypic suppression of Mtr was recognized by reductions of resistance to benzylpenicillin and also to oxacillin and the hydrophobic agents novobiocin and erythromycin. The resistance to each of these antibiotics returned to the Mtr levels in mom+ transformants isolated by selection for increased resistance to either novobiocin or erythromycin; the accompanying change of the outer membrane protein I seroreactions confirmed the proximity of nmp and mom. Thus, some mutant gonococci display wild-type antibiotic susceptibilities but can express multiple resistance following a mom+ mutation that releases the suppressed Mtr phenotype.

Alleles↗
PubMed1997

Amino termini of histones H3 and H4 are required for a1-alpha2 repression in yeast.

The Saccharomyces cerevisiae alpha2 repressor controls two classes of cell-type-specific genes in yeast through association with different partners. alpha2-Mcm1 complexes repress a cell-specific gene expression in haploid alpha cells and diploid a/alpha cells, while a1-alpha2 complexes repress haploid-specific genes in diploid cells. In both cases, repression is mediated through Ssn6-Tu1 corepressor complexes that are recruited via direct interactions with alpha2. We have previously shown that nucleosomes are positioned adjacent to the alpha2-Mcm1 operator under conditions of repression and that Tupl interacts directly with histones H3 and H4. Here, we examine the role of chromatin in a1-alpha2 repression to determine if chromatin is a general feature of repression by Ssn6-Tup1. We find that mutations in the amino terminus of histone H4 cause a 4- to 11-fold derepression of a reporter gene under a1-alpha2 control, while truncation of the H3 amino terminus has a more modest (3-fold or less) effect. Strikingly, combination of the H3 truncation with an H4 mutation causes a 40-fold decrease in repression, clearly indicating a central role for these histones in a1-alpha2-mediated repression. However, in contrast to the ordered positioning of nucleosomes adjacent to the alpha2-Mcm1 operator, nucleosomes are not positioned adjacent to the a1-alpha2 operator in diploid cells. Our data indicate that chromatin is important to Ssn6-Tup1-mediated repression but that the degrees of chromatin organization directed by these proteins differ at different promoters.

Chromatin↗
PubMed1992

Repression of the E. coli lactose operon by cooperation between two individually unproductive "half-operator" sites.

Two remote and weak lac regressor binding sites can be used jointly to repress the synthesis of beta-galactosidase in E. coli, while they cannot separately. When this result is discussed in reference to the various modes of cooperation between the sites, it supports with a new approach a model implying the simultaneous binding of lac repressor to both sites with the formation of a DNA loop. In connection with this point, we present a new strategy to detect cooperative interactions in vivo, based on the asymmetry of the DNA binding site, formally equivalent here to a half-site, heterodimerization of the protein, and influence of orientation of the sites on repression at short and long distance.

DNA, Bacterial↗
PubMed2000

Multiple regulators and their interactions in vivo and in vitro with the cbb regulons of Rhodobacter capsulatus.

The cbb(I) and cbb(II) operons encode structural genes which are important for carbon dioxide fixation via the Calvin-Benson-Bassham reductive pentose phosphate pathway in Rhodobacter capsulatus. Each operon is regulated by cognate LysR-type transcriptional activators, CbbR(I) and CbbR(II), with the product of the cbbR(I) gene, CbbR(I), able to control its own transcription under some growth conditions. Furthermore, CbbR(I) may at least partially regulate the cbb(II) operon, with significant, yet regulated transcription of the cbb(II) operon occurring in the absence of any CbbR. These results suggested the importance of additional regulators. Thus, in addition to the rather specific control exerted by CbbR, a more globally significant regulatory system, the RegA-RegB (PrrA-PrrB) two-component system, was found to contribute to transcriptional regulation of each cbb operon. The regA and regB mutant strains were found to contain constitutive levels of form I and form II RubisCO, the major proteins encoded by the cbb(I) and cbb(II) operons, respectively. In addition, DNaseI footprint analyses indicated that RegA*, a constitutively active mutant form of RegA, binds specifically to cbb(I) and cbb(II) promoter-operator regions. CbbR(I), CbbR(II), and RegA binding loci were localized relative to transcription start sites, leading to a coherent picture of how each of these regulators interacts with specific promoter-operator sequences of the cbb operons.

Bacterial Proteins↗
PubMed1989

Differential repression of GAL4 and adjacent transcription activators by operators in the yeast GAL upstream activating sequence.

The upstream activating sequence of the adjacent and divergently transcribed GAL1 and GAL10 genes of Saccharomyces cerevisiae (UASG) contains at least three distinct classes of overlapping transcriptional control sites. The transcription activator GAL4 binds to four related sites in UASG and induces expression of GAL1 and GAL10 when galactose is available. We showed that UASG contains two additional positive control sites, designated GAL4/galactose-independent activating elements (GAEs), which reside at positions adjacent to or overlapping the GAL4-binding sites. When separated from neighboring sequences in UASG, the GAEs activate transcription independently of GAL4 with no requirement for galactose. In the intact GAL1-GAL10 divergent promoter region, their activity is ordinarily repressed by multiple negative control elements, the GAL operators. When galactose is available, GAL4 overcomes the activity of the GAL operators, while the putative GAE-binding proteins stay repressed. Combined, these results imply that distinct activators (GAL4 and GAE proteins) bound at adjacent or overlapping sites in UASG are differentially regulated by putative repressor proteins simultaneously bound at adjacent GAL operators. We surmise that GAE1 and GAE2 may have a physiological function other than regulation of galactose catabolism per se and discuss three hypotheses to account for their presence in UASG.

Base Sequence↗
PubMed1997

Yeast silencers create domains of nuclease-resistant chromatin in an SIR4-dependent manner.

Previous analysis of the repression of the silent mating type loci in Saccharomyces cerevisiae has linked the mechanism of silencing to the formation of a chromatin domain at the silenced loci. In this study, a TRP1 reporter gene was used to examine changes in chromatin structure in a neutral environment. This enabled the chromatin structure organized by yeast silencers to be compared directly with changes effected by the yeast alpha2 repressor. It was found that silencers mediate the formation of lengthy nuclease-resistant domains on the DNA, rather than specifically positioning nucleosomes over promoter regions as the alpha2 repressor does. Silencing at the TRP1 reporter gene closely resembled silencing at the HMR and HML loci. Repression of the test gene was optimal when two silencers flanking the reporter gene were used, mimicking the situation at the silent loci. In addition, both repression of the reporter gene and the formation of nuclease-resistant chromatin domains was SIR4 dependent.

Aldose-Ketose Isomerases↗
PubMed1998

Mutation analysis of PobR and PcaU, closely related transcriptional activators in acinetobacter.

Acinetobacter PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite p-hydroxybenzoate and activates transcription of pobA, the structural gene for the enzyme that converts p-hydroxybenzoate to protocatechuate. This compound, differing from p-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from Acinetobacter strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which pobR function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the pobA-pobR intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing PcaU to mimic PobR activity were shown to be G222V amino acid substitutions in the C terminus of the 274-residue protein. Together, the analyses suggest that PobR and PcaU possess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.

Acinetobacter↗
PubMed2005

Autorepression of AdpA of the AraC/XylS family, a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus.

AdpA belonging to the AraC/XylS family is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for physiological and morphological differentiation. On the other hand, AdpA repressed its own transcription by cooperative binding to the promoter region containing multiple operator sites. AdpA contained three operator sites, site 1 approximately at nucleotide position -100, site 2 at the promoter elements, and site 3 at position +80. AdpA bound to a strong binding site 1 increased the affinity for AdpA of a weak site 2, probably by forming a DNA loop via the two molecules of AdpA dimer, thus preventing RNA polymerase from access to the promoter. AdpA bound to site 3 with rather weak affinity repressed the AdpA promoter activity independently of sites 1 and 2, perhaps preventing RNA polymerase from chain elongation. Consistent with this model, the in vivo transcription of AdpA containing mutated site 1 or site 3 was greatly increased. In addition, streptomycin production, one of the phenotypes controlled positively by AdpA, was greatly increased in the mutants containing AdpA with a mutation at site 1 and site 3. The in vitro transcription of AdpA containing mutated site 1 was also increased. Thus, the transcription of AdpA, encoding an important transcriptional factor for ordered physiological and morphological development, is self-controlled.

Bacterial Proteins↗
PubMed1997

A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae.

A set of Saccharomyces cerevisiae expression vectors has been developed in which transcription is driven by a hybrid tetO-CYC1 promoter through the action of a tetR-VP16 (tTA) activator. Expression from the promoter is regulated by tetracycline or derivatives. Various modalities of promoter and activator are used in order to achieve different levels of maximal expression. In the presence of antibiotic in the growth medium at concentrations that do not affect cell growth, expression from the tetO promoter is negligible, and upon antibiotic removal induction ratios of up to 1000-fold are observed with a lacZ reporter system. With the strongest system, overexpression levels comparable with those observed with GAL1-driven promoters are reached. For each particular promoter/tTA combination, expression can be modulated by changing the tetracycline concentration in the growth medium. These vectors may be useful for the study of the function of essential genes in yeast, as well as for phenotypic analysis of genes in overexpression conditions, without restrictions imposed by growth medium composition.

Base Sequence↗
PubMed2006

Propionate-regulated high-yield protein production in Escherichia coli.

A new expression system containing the Salmonella enterica prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prp BCDE) and prpR encoding the positive regulator of this promoter has been developed and tested. The main features of the expression system compared to those based on the bacteriophage T7 promoter are low background expression and high induced expression in Escherichia coli strains BL21, BL21(DE3), MG1655, and W3110. In addition, propionate is an inexpensive, simple-to-use, nontoxic inducer that is attractive for large-scale protein production. Hence, this new system is highly complementary to the widely used T7 promoter-driven expression systems.

Bacteriophage T7↗