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At least 19 recordsLinked to original sources
PubMed1992

The Streptomyces glaucescens TcmR protein represses transcription of the divergently oriented tcmR and tcmA genes by binding to an intergenic operator region.

Preliminary evidence has been presented by Guilfoile and Hutchinson (J. Bacteriol. 174:3651-3658, 1992) suggesting that the Streptomyces glaucescens TcmR protein is a transcriptional repressor. Here, we extend that work by showing that transcription of the S. glaucescens tcmA gene is inducible by tetracenomycin C and that inactivation of the tcmR gene results in constitutive transcription of the tcmA gene. Gel retardation studies show that the TcmR protein binds to the tcmA-tcmR intergenic region in vitro and that this binding is inhibited by tetracenomycin C. Footprinting experiments demonstrate that the TcmR protein binds to an operator region that encompasses both the tcmA and the tcmR promoters. This genetic and biochemical evidence strongly supports the model of the TcmR protein acting as a repressor in inhibiting transcription of both the tcmA and the tcmR genes, in much the same way that TetR from Tn10 inhibits transcription of tetA and tetR.

Amino Acid Sequence↗
PubMed1996

Regulation of gene expression at a distance: the hypothetical role of regulatory protein-mediated topological changes of DNA.

A theoretical model is presented that a regulatory protein may activate the transcription of a promoter by interacting with a single remote operator. In response to an inducer molecule the regulatory protein bound to the operator undergoes a conformational change, and might mediate a B to Z-DNA conversion of the operator. This transition would remove both helical turns and supercoils from the intervening region between the operator and the promoter, resulting in the correct spatial arrangement of the -10 and -35 hexamers of the promoter, which therefore can be efficiently transcribed.

Agrobacterium tumefaciens↗
PubMed1993

A linguistic representation of the regulation of transcription initiation. I. An ordered array of complex symbols with distinctive features.

The inadequacy of context-free grammars in the description of regulatory information contained in DNA gave the formal justification for a linguistic approach to the study of gene regulation. Based on that result, we have initiated a linguistic formalization of the regulatory arrays of 107 sigma 70 E. coli promoters. The complete sequences of promoter (Pr), operator (Op) and activator binding sites (I) have previously been identified as the smallest elements, or categories, for a combinatorial analysis of the range of transcription initiation of sigma 70 promoters. These categories are conceptually equivalent to phonemes of natural language. Several features associated with these categories are required in a complete description of regulatory arrays of promoters. We have to select the best way to describe the properties that are pertinent for the description of such regulatory regions. In this paper we define distinctive features of regulatory regions based on the following criteria: identification of subclasses of substitutable elements, simplicity, selection of the most directly related information, and distinction of one array among the whole set of promoters. Alternative ways to represent distances in between regulatory sites are discussed, permitting, together with a principle of precedence, the identification of an ordered set of complex symbols as a unique representation for a promoter and its associated regulatory sites. In the accompanying paper additional distinctive features of promoters and regulatory sites are identified.

Binding Sites↗
PubMed2000

Interaction of CbbR and RegA* transcription regulators with the Rhodobacter sphaeroides cbbIPromoter-operator region.

The form I (cbb(I)) Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle operon of Rhodobacter sphaeroides is regulated by both the transcriptional activator CbbR and the RegA/PrrA (RegB/PrrB) two-component signal transduction system. DNase I footprint analyses indicated that R. sphaeroides CbbR binds to the cbb(I) promoter between -10 and -70 base pairs (bp) relative to the cbb(I) transcription start. A cosmid carrying the R. capsulatus reg locus was capable of complementing an R. sphaeroides regA-deficient mutant to phototrophic growth with restored regulated synthesis of both photopigments and ribulose-bisphosphate carboxylase/oxygenase (Rubisco). DNase I footprint analyses, using R. capsulatus RegA*, a constitutively active mutant version of RegA, detected four RegA* binding sites within the cbb(I) promoter. Two sites were found within a previously identified cbb(I) promoter proximal regulatory region from -61 to -110 bp. One of these proximal RegA* binding sites overlapped that of CbbR. Two sites were within a previously identified promoter distal positive regulatory region between -301 and -415 bp. Expression from promoter insertion mutants showed that the function of the promoter distal regulatory region was helical phase-dependent. These results indicated that RegA exerts its regulatory affect on cbb(I) expression through direct interaction with the cbb(I) promoter.

Bacterial Proteins↗
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↗
PubMed1993

A linguistic representation of the regulation of transcription initiation. II. Distinctive features of sigma 70 promoters and their regulatory binding sites.

The goal of this paper and the accompanying one is to achieve a linguistic representation of a set of sigma 70 promoters. Such a description is formed by an ordered concatenated array of complex symbols identified by their categorical property, i.e. promoter, operator, activator binding site, etc. Each of these symbols may contain several properties associated with their respective classes of 'molecular words'. The main problem in attaining such a description is to define which properties are going to be represented, and how. In the accompanying paper the criteria on which the selection of alternative descriptions is based were discussed. The properties of promoters and regulatory sites are discussed here, and their corresponding distinctive features are selected following such criteria. Thus, information that is not directly relevant and that can overspecify the description has been excluded, since it does not seem to contribute to identifying classes of substitutable elements. Other properties, such as strength of promoters, position of regulatory sites, different types of specificities of regulatory proteins, affinity of their binding sites, etc., are also discussed. As a result of this analysis, a complete representation with distinctive features of the set of sigma 70 promoters is attainable.

Binding Sites↗
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↗
PubMed2002

Genetic organization of the lexA, recA and recX genes in Xanthomonas campestris.

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.

Bacterial Proteins↗
PubMed1994

Multiple positive and negative cis-acting elements of the STA2 gene regulate glucoamylase synthesis in Saccharomyces cerevisiae.

Expression of the glucoamylase-encoding gene (STA2) in Saccharomyces cerevisiae was previously shown to be regulated transcriptionally by both positive and negative factors. The objective of this work was to identify the cis-acting elements responsible for STA2 transcriptional activation as well as the transcriptional repressor effects of STA10 and MATa/MAT alpha. We identified two upstream activation regions (UAS). Three repressor regions responsive to STA10-mediated repression were identified, as well as two regions for down-regulation of STA2 expression. MATa/MAT alpha repression appears to effect STA2 expression either downstream from the translational start site or, indirectly, since no functional a1/alpha 2-responsive sequence was identified in the promoter region.

Base Sequence↗
PubMed1988

The inducible lac operator-repressor system is functional for control of expression of injected DNA in Xenopus oocytes.

We have investigated the use of the Escherichia coli lac operator-repressor system to regulate the expression of genes introduced by microinjection into Xenopus laevis oocytes. We observe that expression of an MSV-cat fusion gene, in which the lac operator was inserted between the TATA box and the transcription start point (tsp), or between the tsp and the start codon (ATG), is completely repressed when the lac repressor protein is added to the plasmid suspension prior to injection. The lac repressor had no detectable effect on the expression of a coinjected HSV-1 tk gene that had no operator insertion (or on an MSV-cat gene without an operator), indicating that the nonspecific DNA-binding properties of the repressor do not inhibit transcription. CAT activity expressed from the operator-containing MSV-cat genes transcribed in the oocyte nucleus was also inhibited by repressor injected into the oocyte cytoplasm, showing that biologically active repressor proteins can enter the nucleus from the cytoplasm. Injection of the inducer IPTG into the oocyte cytoplasm markedly derepressed the repressed cat genes but not the HSV-1 tk gene coinjected as an internal control. Overall, our results show that the lac operator-repressor system can be useful as a genetic switch in the regulation of gene expression of injected DNA in frog oocytes. Finally, our observations on the vectors used in this work show that the MSV enhancer significantly activates transcription from the SV40 early promoter in frog oocytes, although previous studies have indicated that the MSV enhancer is not necessary for the activity of the MSV promoter in oocytes [Graves et al., Mol. Cell. Biol. 5 (1985) 1945-1958].

Animals↗
PubMed1988

The Escherichia coli LexA repressor-operator system works in mammalian cells.

We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells. The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40. This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein. We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene. Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter. Necessary 3' signals were from the HSV tk gene. Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor. We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.

Animals↗
PubMed1987

Nucleotide sequence and expression of the pyrC gene of Escherichia coli K-12.

The pyrC gene of Escherichia coli K-12, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, was cloned as part of a 1.6-kilobase-pair chromosomal fragment. The nucleotide sequence of this fragment was determined. An open reading frame encoding a 348-amino acid polypeptide (Mr = 38,827) was identified as the pyrC structural gene by comparing the amino acid composition predicted from the DNA sequence with that previously determined for the dihydroorotase subunit. The pyrC promoter was mapped by primer extension of in vivo transcripts. Transcriptional initiation was shown to occur within a region located 36 to 39 base pairs upstream of the pyrC structural gene. Pyrimidine availability appears to affect the use of the minor transcriptional initiation sites. The level of pyrC transcription and dihydroorotase synthesis was coordinately derepressed by pyrimidine limitation, indicating that regulation occurs, at least primarily, at the transcriptional level. Inspection of the pyrC nucleotide sequence indicates that gene expression is not regulated by an attenuation control mechanism similar to that described for the pyrBI operon and the pyrE gene. A possible mechanism of transcriptional control involving a common repressor protein is suggested by the identification of a highly conserved, operatorlike sequence in the promoter regions of pyrC and the other pyrimidine genes (i.e., pyrD and carAB) whose expression is negatively regulated by a cytidine nucleotide effector.

Amidohydrolases↗
PubMed2000

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

Binding Sites↗
PubMed1997

Regulation by tetracycline of gene expression in Saccharomyces cerevisiae.

A convenient system for the control of gene expression in Saccharomyces cerevisiae was developed. Tetracycline-responsive promoters were constructed by fusing the tetracycline operator (tetO) to the S. cerevisiae HOP1 promoter. When fused to the tetracycline repressor (tetR), trans-activation domains of both GAL4 and HAP4 were capable of promoting transcription from the tetO-HOP1 chimeric promoter, but the tetR-HAP4 fusion activator was the more efficient transcriptional activator. Addition of tetracycline nearly completely repressed activator-dependent transcription from the tetO-HOP1 promoter. Moreover, tetracycline-dependent repression of YEF3, CDC28 and RAM2 expression impaired cell growth. Thus, this system is useful for the elucidation of gene function in S. cerevisiae.

Gene Expression Regulation, Fungal↗
PubMed1997

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism.

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent Km of 6 microM for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have - 10 and -35 sequence elements typical of sigma70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 microM gluconate, modestly induced by very low levels of gluconate (4 microM), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

Bacterial Proteins↗
PubMed1993

Relief of triple-helix-mediated promoter inhibition by elongating RNA polymerases.

We have characterized triple-helix-mediated inhibition of an artificial bacteriophage promoter with respect to relief of inhibition by incoming RNA polymerases that initiate upstream or downstream from the operator sequence. Whereas oligonucleotide-directed triple-helix formation inhibits the test promoter, promoter activity is restored when the triple-helical complexes are disrupted by transcription of either strand of the homopurine operator sequence. The degree of relief from inhibition is related to the frequency of operator transcription. These observations demonstrate that this artificial repressor-operator complex is subject to antagonism by cis elements (other promoters) acting at a distance. Such antagonism might also arise between certain natural transcriptional control regions. Our results suggest that the efficiency of artificial repressors based on triple-helix formation may be limited by transcriptional activity in the gene control region.

Bacteriophage T7↗
PubMed1987

Expression of ricin A chain in Escherichia coli.

DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3. Transcription is controlled from the coliphage promoter PN25 fused with the lac operator of E.coli. When induced, E.coli 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity.

DNA↗