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At least 361 records · Page 20Linked to original sources
PubMed1995

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Bacillus subtilis↗
PubMed1995

Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins.

Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for expressing a thyrotropin (TSH) receptor capable of ligand and Graves' disease autoantibody binding comparable to the receptor produced in mammalian cells. Because of the clinical importance of high level expression of this protein, we reassessed the baculovirus system using a new transfer vector (pAcMP3) containing the late basic protein promoter, which functions earlier than the classical polyhedrin promoter. Maximal synthesis of the [35S]methionine-labeled TSH receptor extracellular domain, affinity-purified using a 6-histidine tag, occurred earlier (1 day after insect cell infection) than with a vector (pVL1393) containing the polyhedrin promoter. The pAcMP3-derived TSH receptor extracellular domain was larger (approximately 68 kDa) than the pVL1393-derived protein (approximately 63 kDa). Only the 68-kDa product was secreted, albeit in trace amounts detectable only by precursor labeling. Enzymatic deglycosylation reduced both 68- and 63-kDa cellular proteins to approximately 54 kDa, indicating that the pAcMP3 vector generated a protein with greater carbohydrate content. However, despite its greater degree of glycosylation, most of the 68-kDa protein remained within the cell, almost entirely in the particulate fraction. Remarkably, the trace amounts of 68-kDa receptor protein affinity-purified from the soluble cytosolic fraction of infected insect cells completely neutralized TSH receptor autoantibodies in patients' sera and partly inhibited TSH binding. In conclusion, a baculovirus vector with a promoter active earlier than the conventional polyhedrin promoter generates a more glycosylated and functional TSH receptor extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of functional, highly glycosylated proteins.

Animals↗
PubMed1996

A GC-rich region containing Sp1 and Sp1-like binding sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes. Implication In the overactivity of FAS promoter in obese Zucker rats.

We have previously shown that the proximal 2-kb sequence of the fatty acid synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/fa) versus lean Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter activity mainly depends on a region from -200 to -126. This sequence exerts a strong negative effect on FAS promoter in adipocytes from lean rats but not in those from obese rats, resulting in a marked overtranscriptional activity in the latter cells. This region, fused to a heterologous promoter, the E1b TATA box, induced differential levels of gene reporter activity in lean and obese rat adipocytes, indicating it harbors fa-responsive element(s). Whatever the rat genotype, adipocyte nuclear proteins were shown to footprint the same protected sequence within the fa-responsive region, and supershift analysis demonstrated that Sp1 or Sp1-like proteins were bound to this DNA subregion. Compelling evidence that the Sp1 binding site contained in this sequence was implicated in the differential promoter activity in lean versus obese rats, was provided by the observation that mutations at this Sp1 site induced a 2.5-fold increase in FAS promoter activity in adipocytes from lean rats, whereas they had no effect in adipocytes from obese rats.

Adipocytes↗
PubMed1998

Hypoxia-inducible mammalian gene expression analyzed in vivo at a TATA-driven promoter and at an initiator-driven promoter.

We have analyzed protein-DNA interactions in vivo at transcriptional control elements for two hypoxia-inducible genes in mouse hepatoma cells. The promoter for the phosphoglycerate kinase 1 (PGK1) gene contains an initiator element, but no TATA sequence, whereas the promoter for the glucose transporter 1 (Glut1) gene contains a TATA element but no initiator sequence. Our findings reveal hypoxia-inducible, Arnt-dependent occupancy of DNA recognition sites for hypoxia-inducible factor 1 (HIF-1) upstream of both target genes. The conserved recognition motif among the five recognition sites is 5'-CGTG-3'. The PGK1 promoter exhibits constitutive occupancy of a binding site for an unknown protein(s); however, we detect no protein-DNA interaction at the initiator element, in either uninduced or induced cells. The Glut1 promoter also exhibits constitutive protein binding; in addition, the TATA element exhibits partial occupancy in uninduced cells and increased occupancy under hypoxic conditions. We find no evidence for hypoxia-induced changes in chromatin structure of either gene. Time-course analyses of the Glut1 gene reveal a temporal relationship between occupancy of HIF-1 sites and TATA element occupancy. Our findings suggest that the promoters for both hypoxia-responsive genes constitutively maintain an accessible chromatin configuration and that HIF-1 facilitates transcription by recruiting and/or stabilizing a transcription factor(s), such as TFIID, at both promoters.

Animals↗
PubMed2004

Oct-1 maintains an intermediate, stable state of HLA-DRA promoter repression in Rb-defective cells: an Oct-1-containing repressosome that prevents NF-Y binding to the HLA-DRA promoter.

The cell surface HLA-DR molecule binds foreign peptide antigen and forms an intercellular complex with the T cell receptor in the course of the development of an immune response against or immune tolerance to the antigen represented by the bound peptide. The HLA-DR molecule also functions as a receptor that mediates cell signaling pathways, including as yet poorly characterized pathway(s) leading to apoptosis. Expression of HLA-DR mRNA and protein is ordinarily inducible by interferon-gamma but is not inducible in tumor cells defective for the retinoblastoma tumor suppressor protein (Rb). In the case of the HLA-DRA gene, which encodes the HLA-DR heavy chain, previous work has indicated that this loss of inducibility is attributable to Oct-1 binding to the HLA-DRA promoter. In this report, we used Oct-1 antisense transformants to determine that Oct-1 represses the interferon-gamma response of the endogenous HLA-DRA gene. This determination is consistent with results from a chromatin immunoprecipitation assay, indicating that Oct-1 occupies the endogenous HLA-DRA promoter when the HLA-DRA promoter is inactive in Rb-defective cells but not when the promoter is converted to a previously defined, transcriptionally competent state, induced by treatment of the Rb-defective cells with the HDAC inhibitor, trichostatin A. In vitro DNA-protein binding analyses indicated that Oct-1 prevents HLA-DRA promoter activation by mediating the formation of a complex of proteins, termed DRAN (DRA negative), that blocks NF-Y access to the promoter.

Binding Sites↗
PubMed2006

A consensus adenine at position -11 of the nontemplate strand of bacterial promoter is important for nucleation of promoter melting.

Numerous studies have suggested an important role of adenine at position -11 of the nontemplate strand of bacterial promoters for sequence-specific recognition of the -10 promoter element in single-stranded form. In this work, we attempted to identify a specific step in transcription initiation reaction that is most critically dependent on specific recognition of -11A. Mutating -11A in the context of a model promoter resulted in a profound decrease of the rate of heparin-resistant promoter complex formation and in a modest increase of the rate of heparin-resistant complex dissociation. The identity of nontemplate base at position -11 became relatively unimportant when the duplex in the vicinity of this position was destabilized by base pair mismatches. For promoters with a nonnative thymine at nontemplate position -11, we observed a remarkable correlation between the rate of heparin-resistant complex formation (or transcription activity) and the free energy of duplex stability in the vicinity of this residue, indicating that the replacement of -11A with a T affected a step in the reaction that involves local melting of DNA duplex. These data show that a promoter melting defect caused by a loss of RNA polymerase contact with -11A can be rescued by artificially induced local destabilization of the DNA duplex. These results are consistent with and support the idea that specific recognition of adenine at the nontemplate -11-position is important only for the initial nucleation of melting, which probably involves the flipping of this adenine out from the DNA duplex.

Adenine↗
PubMed1991

Prolactin-deficient GH3B3 cells are defective in the utilization of the endogenous prolactin promoter yet are fully competent to initiate transcription from a transfected prolactin promoter.

Transcription of the prolactin (PRL) gene has been analyzed in wild-type D6, PRL-deficient B3, and revertant r16 GH3 cells. Levels of processed nuclear transcripts from the PRL gene were substantially reduced in the deficient line compared to wild-type cells and returned to greater than wild-type levels in the revertant line. Rare PRL transcripts in the deficient line contained the same 5' end found on transcripts in wild-type and revertant cells as judged by primer extension and S1 nuclease protection assays, implying that the cells are deficient in utilization of the normal wild-type promoter. Deficient cells also contained wild-type levels of the PRL- and growth hormone-specific transcription factor pit-1/GHF-1, and no difference was found in the ability of extracts from wild-type and deficient cells to retard various restriction fragments from both the proximal and the distal PRL promoter regions. The deficient and wild-type cells were equally competent in initiating transcription from a transfected rat PRL promoter containing both the distal and proximal promoter elements. These observations imply that PRL-deficient cells are not defective in a trans-activating factor functioning on these PRL promoter fragments (trans model). Rather, inefficient use of the PRL promoter in the variant cells may reflect an increased methylation state of the PRL gene itself (cis model).

Animals↗
PubMed2006

Dragon Promoter Mapper (DPM): a Bayesian framework for modelling promoter structures.

UNLABELLED: Dragon Promoter Mapper (DPM) is a tool to model promoter structure of co-regulated genes using methodology of Bayesian networks. DPM exploits an exhaustive set of motif features (such as motif, its strand, the order of motif occurrence and mutual distance between the adjacent motifs) and generates models from the target promoter sequences, which may be used to (1) detect regions in a genomic sequence which are similar to the target promoters or (2) to classify other promoters as similar or not to the target promoter group. DPM can also be used for modelling of enhancers and silencers. AVAILABILITY: http://defiant.i2r.a-star.edu.sg/projects/BayesPromoter/ CONTACT: vlad@sanbi.ac.za SUPPLEMENTARY INFORMATION: Manual for using DPM web server is provided at http://defiant.i2r.a-star.edu.sg/projects/BayesPromoter/html/manual/manual.htm.

Algorithms↗
PubMed1998

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Bacterial Proteins↗
PubMed2005

Molecular basis for promoter selectivity of the transcriptional activator OmpR of Escherichia coli: isolation of mutants that can activate the non-cognate kdpABC promoter.

Escherichia coli has two osmo-responsive two-component regulatory systems, the EnvZ-OmpR and KdpD-KdpE systems, each of which consists of a sensor histidine protein kinase and a response regulator. The OmpR and KdpE response regulators belong to the same family of DNA-binding proteins, and act as positive transcriptional factors in response to the medium osmolarity. However, OmpR specifically activates the ompC gene encoding the OmpC outer membrane protein, whereas KdpE exclusively activates the kdpABC operon encoding the high-affinity Kdp potassium-transporter. To gain insight into the molecular basis for such strict promoter selectivity, we isolated OmpR mutants that can activate the non-cognate kdpABC promoter in vivo. For these OmpR mutants, it was found that a few common and crucial amino acids are responsible for the altered property of OmpR (e.g., Gly-164, Glu-193). In vitro properties of these OmpR mutants were further examined by means of DNA-binding assays and DNA-footprinting analyses with reference to the kdpABC promoter. These results were interpreted on the basis of the three-dimensional structure of the C-terminal half of OmpR, which consists of a DNA-binding helix-turn-helix motif and a RNA polymerase-interacting surface. The results of this study were best explained by assuming that the isolated OmpR mutants have an altered property with regard to the interaction with RNA polymerase on the kdpABC promoter. We propose that the promoter selectivity of OmpR is determined not only by its DNA-binding specificity, but also by the spatial configuration of the promoter on which OmpR must properly associate with RNA polymerase.

Adenosine Triphosphatases↗
PubMed1985

The mac promoters: functional hybrid promoters activated by the malT product and repressed by the lacI product.

Using in vitro techniques we have fused upstream sequences from the malPp promoter (normally activated by the MalT protein) to downstream sequences from the lacZp promoter (normally repressed by the LacI protein). Several hybrid promoters were thus obtained, which were controlled by the MalT protein, but were poorly active. More efficient promoters were then isolated using in vivo selection. Three main conclusions could be derived from the analysis of all of these hybrid promoters. Firstly, the MalT protein seems able to force RNA polymerase to start transcription at any DNA sequence, albeit with a low efficiency. Secondly, the strength of the hybrid promoters is considerably increased if a Pribnow Box is positioned at a precise location with respect to the MalT binding site. Thirdly, the presence of the lac operator, even when properly positioned with respect to the transcription startpoint, does not suffice to permit full repression by the lacI product.

Base Sequence↗
PubMed1986

Interaction between E. coli RNA polymerase and the tetR promoter from pSC101: homologies and differences with other E. coli promoter systems from close contact point studies.

The interaction between E. coli RNA polymerase and the tetR promoter from pSC101, was studied by protection and premodification experiments, using dimethyl sulfate, methylation of single stranded cytosines, and DNAase I footprinting. Whereas qualitative and quantitative results from the chemical approach conform to patterns already displayed by other promoter systems, hypersensitive sites to DNAase I attack differ from those of other promoters. Distribution and nature of the contacts suggest that regions of the promoter sequence participates differently in complex formation. The involvement of major and minor grooves of the double helix in the complex with the enzyme, differs along the promoter. After a comparison of the results from seven different promoters, a pattern of conserved contacts seem to appear. Comparison of temperature dependence of local unwinding around the transcription start site (detected by the appearance of single stranded cytosines), and DNAase I footprinting, reveals that the process leading to stable complex formation can be achieved without disruption of base-pairing.

Base Sequence↗
PubMed1989

A novel method for promoter search enhanced by function-specific subgrouping of promoters--developed and tested on E.coli system.

A new method for evaluating some complex characteristics of the primary structure of E.coli promoters is proposed. The method, of nonparametric statistical significance, selects important conserved single-base positions in combination with 2-base coupling relations of identity and complementarity. The extended consensus of promoter characteristics thus obtained was used to scan unknown sequences for similarity with E.coli promoters. In terms of this method, a complete set of 244 E.coli promoters was shown to be structurally inconsistent. The set was then broken down into functionally homogeneous subsets of promoters to enhance the selectivity of the search for E.coli-specific promoter sequences, with a high significance level being attained.

Algorithms↗
PubMed1990

Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR.

We have made site-specific mutations in the promoter-operator region of the mercury-resistance (mer) operon of transposon Tn501. Mutations were selected to alter the spacing between the -10 and -35 promoter elements without altering the sequence of a 7 bp dyad symmetrical sequence, which is the site of binding of the regulatory protein, MerR. (MerR acts as a repressor in the absence of mercuric salts and as an inducer in their presence, and binds to the same site in each case). Transcription from the mutant promoters was measured in vivo in the presence and absence of MerR and of mercuric salts; and the relative affinities of the mutant promoters for partially purified MerR were determined in vitro by gel-shift assay in the presence and absence of mercuric salts. The 19 bp spacer was found to be essential for correct induction and repression of the operon; a spacer size of 20 or 21 bp prevents induction, and a spacer size of 18 or 17 bp causes the promoter to be highly active under all conditions. Double mutations, which alter the position of the 7 bp dyad relative to the -10 and -35 sequences without altering their spacing prevent induction by the MerR-Hg(II) complex, demonstrating the tight constraints on the positions of MerR and RNA polymerase in the transcriptional complex. The data are compatible with a model for induction of the mer promoter involving a local conformational change in the DNA structure caused by the MerR-Hg(II) complex.

Bacterial Proteins↗
PubMed1990

Regulation of a human neurotropic virus promoter, JCVE: identification of a novel activator domain located upstream from the 98 bp enhancer promoter region.

Transcription of the human neurotropic virus promoter, JCVE, and its regulation in glial cells are controlled by the 98 bp tandem repeats positioned between the viral early and late genes. Here, we show that a region, designated domain-D, located upstream from the 98 bp repeats functions as a transcriptional activator and increases JCVE promoter activity. Using the reporter SV40E promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, we demonstrate that domain-D stimulates the basal SV40E promoter activity in glial and to a lesser degree in HeLa cells. Results from gel mobility-shift assays indicate that domain-D interacts with proteins derived from glial and HeLa extracts and results in the formation of specific DNA-protein complexes. Through UV cross-linking assays, we demonstrate that these complexes have similar electrophoretic mobilities which comigrate with the 43-50 Kd proteins derived from glial and HeLa cells. These findings, together with our previous observations, imply that the JCVE control region is composed of multiple common and specific activator domains that may account for the increased expression of the promoter in glial cells. The possible role of the D-binding protein in transcription of the JCVE promoter is discussed.

Animals↗
PubMed1992

Escherichia coli promoters: neural networks develop distinct descriptions in learning to search for promoters of different spacing classes.

Back-propagation neural networks were trained to recognize promoter sequences of each of the three major spacing classes found in E. coli. These networks were trained with the object of maximizing their ability to generalize while maintaining the level of false positive identifications at a fraction of 1 percent. These objectives were generally met. Networks for the 16 base spacing class captured between 78 and 100% of previously unseen promoters in different tests; networks for the 17 base class identified 97% of the test promoters; networks for the 18 base class identified 79% of the test promoters. A tandem poll of networks for all three spacing classes produced a cumulative false positive level of less than 0.5%. In each case, the weight matrices used by the networks in their classification were analyzed to determine the relative weight assigned to the occurrence of a given base at a given position within the promoter. In this fashion, an approximate description of the network's definition of the promoter can be obtained.

Base Sequence↗
PubMed1995

Identification of close contacts between the sigma N (sigma 54) protein and promoter DNA in closed promoter complexes.

The complexes forming between the alternative sigma factor protein sigma N (sigma 54), its holoenzyme and promoter DNA were analysed using the hydroxyl radical probe and by photochemical footprinting of bromouridine-substituted DNA. Close contacts between the promoter, sigma N and its holoenzyme appear to be restricted predominantly to one face of the DNA helix, extending from -31 to -5. They all appear attributable to sigma N and no extra close contacts from the core RNA polymerase subunits in the holoenzyme-promoter DNA complex were detected. We suggest that the apparent absence of close core RNA polymerase contacts in the region of the promoter DNA to be melted during open complex formation is important for maintaining the closed complex. Results of the hydroxyl radical footprinting imply that sigma N makes multiple DNA backbone contacts across and beyond the -12, -24 consensus promoter elements, and the photochemical footprints indicate that consensus thymidine residues contribute important major groove contacts to sigma N. Formation of the open complex is shown to involve a major structural transition in the DNA contacted by sigma N, establishing a direct role for sigma N in formation of the activated promoter complex.

Base Sequence↗
PubMed1996

Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in Pm identifies a new region required for promoter function.

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase holoenzyme and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a recognizable -10 region but lacks a -35 region. Mutagenesis of this promoter (from -70 to +10) was performed using mutagenic oligonucleotide-directed PCR. The resulting fragments were cloned into a promoter-lacZfusion vector and analyzed for promoter activity by assaying beta-galactosidase production. Single point mutations with a Down phenotype were clustered in three regions: the -10 region, the Mor footprint region and the spacer between them. Gel retardation experiments with purified Mor protein and promoter mutants demonstrated that sequences important for Mor binding are located within the Mor footprint region and lead us to propose the existence of a dyad symmetry element involved in Mor binding. In agreement with this prediction, glutaraldehyde crosslinking of Mor in solution generated a species with the size of a dimer. These experiments also identified an unusual group of mutations located in the spacer region adjacent to the Mor footprint. These mutations alter promoter activity without affecting Mor binding. A circular permutation assay revealed that Mor does not introduce a significant bend upon binding to its target sequence.

Bacteriophage mu↗