Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retinoid X receptors (RXRs) are recently characterized transcription factors that are members of the nuclear hormone receptor superfamily. However, it is not known whether the endogenous RXR complex requires its ligand for access to its hormone response element (HRE) of a target gene in vivo. Hence, dimethyl sulfate-based genomic footprinting was carried out to examine occupancy of HREs in the retinoic acid (RA) receptor beta2 (RARbeta2) gene promoter in the murine melanoma cell line S91 cultured in the absence or presence of T3, all-trans-RA (atRA), or CD2624, an RXR-selective retinoid. No footprint was observed at the RA-response element (betaRARE) in the absence of ligands. However, a footprint was detected at the betaRARE and other cis-acting elements after a 6 h incubation with CD2624 and atRA. Interestingly, only the betaRARE was footprinted after 60 min incubation with CD2624. These results suggest that the endogenous RXR complex can interact with an HRE of a target gene in the presence of ligand, and subsequently may initiate additional interactions between DNA and other transcription factors.
Mol Cell Endocrinol 1998 Jan 15
PMID:Ligand-inducible retinoid X receptor-mediated protein: DNA interactions in the retinoic acid receptor beta2 gene promoter in vivo. 954 14

In the ciliated protozoan Tetrahymena thermophila the ribosomal DNA (rDNA) minichromosome replicates partially under cell cycle control and is also subject to a copy number control mechanism. The relationship between rDNA replication and rRNA gene transcription was investigated by the analysis of replication, transcription, and DNA-protein interactions in a mutant rDNA, the rmm3 rDNA. The rmm3 (for rDNA maturation or maintenance mutant 3) rDNA contains a single-base deletion in the rRNA promoter region, in a phylogenetically conserved sequence element that is repeated in the replication origin region of the rDNA minichromosome. The multicopy rmm3 rDNA minichromosome has a maintenance defect in the presence of a competing rDNA allele in heterozygous cells. No difference in the level of rRNA transcription was found between wild-type and rmm3 strains. However, rmm3 rDNA replicating intermediates exhibited an enhanced pause in the region of the replication origin, roughly 750 bp upstream from the rmm3 mutation. In footprinting of isolated nuclei, the rmm3 rDNA lacked the wild-type dimethyl sulfate (DMS) footprint in the promoter region adjacent to the base change. In addition, a DMS footprint in the origin region was lost in the rmm3 rDNA minichromosome. This is the first reported correlation in this system between an rDNA minichromosome maintenance defect and an altered footprint in the origin region. Our results suggest that a promoter region mutation can affect replication without detectably affecting transcription. We propose a model in which interactions between promoter and origin region complexes facilitate replication and maintenance of the Tetrahymena rDNA minichromosome.
Mol Cell Biol 1998 May
PMID:A promoter region mutation affecting replication of the Tetrahymena ribosomal DNA minichromosome. 956 21

The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity. These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.
Mol Cell Biol 1998 May
PMID:Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae. 959 2

The Escherichia coli biotin repressor is a member of the "winged helix-turn-helix" class of site-specific DNA binding proteins. The protein binds as a dimer to the 40 bp biotin operator sequence. Although the structure of the aporepressor has been solved by X-ray crystallographic techniques, no structure of the holorepressor-DNA complex is yet available. In order to characterize the structural features of the biotin repressor-biotin operator interface we have applied a number of solution techniques including DNase I, hydroxyl radical and dimethyl sulfate footprinting and the circular permutation or "bending" assay. Results of these combined studies indicate that each repressor monomer forms a bipartite interface with each half-site of the biotin operator sequence. The results imply that, in addition to the helix-turn-helix module of each monomer, a second structural element participates in the protein-DNA interface. The two bipartite protein-DNA interfaces appear, moreover, to primarily involve the two 12 bp termini of the operator site. Results of combined DNase I footprinting and circular permutation analysis indicate, furthermore, that the central 16 bp region that links the two termini becomes distorted concomitant with binding of holoBirA.
J Mol Biol 1998 May 15
PMID:A map of the biotin repressor-biotin operator interface: binding of a winged helix-turn-helix protein dimer to a forty base-pair site. 961 42

The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1, 10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.
Mol Cell Biol 1998 Aug
PMID:Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences. 967 77

ACTH production by non-pituitary tumors is generally not suppressible by exogenous glucocorticoid administration. We had postulated that defects in the glucocorticoid receptor (GR) signaling system might be responsible for this apparent glucocorticoid resistance and had previously demonstrated that DMS-79 cells, derived from an ectopic ACTH-producing tumor, express an abnormal GR mRNA. In this DMS-79 cell GR the sequence normally derived from exons 8 and 9 is replaced by sequence unmatched in the DNA databases. The protein encoded by this mRNA lacks the steroid-binding domain and does not function as a ligand-activated transcription factor. In the present work, we sought to identify the origin of the novel GR mRNA sequence. Southern blot analysis of DMS-79 genomic DNA showed no major structural alteration of the GR gene. Southern blotting of cosmid clones of the normal GR gene revealed that the novel DMS-79 GR mRNA sequence is derived from intron G, between exons 7 and 8. No splice site mutations were found in PCR-amplified DMS-79 DNA fragments surrounding the downstream splice junctions. Further sequencing indicated that the aberrant GR transcript appears to be generated by use of a consensus cleavage/polyadenylation signal found 3650 base pairs into the normal intron G. We conclude that abnormal GR pre-mRNA processing rather than a GR gene mutation confers glucocorticoid resistance on DMS-79 cells.
Mol Cell Endocrinol 1998 Jul 25
PMID:An ACTH-producing small cell lung cancer expresses aberrant glucocorticoid receptor transcripts from a normal gene. 978 13

Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active assemblies. Characterization of population distributions and structural characteristics of intermediate and product conformations is necessary to understand both the mechanisms and the thermodynamics of these processes. For these purposes, here we develop the quantitative method of multiple hit footprinting (MHF), where chemical or enzymatic probing is performed as a function of either concentrations of the footprinting agent and/or time of exposure to it, in the multiple hit regime where many of the population or subpopulation of reactive DNA molecules are modified at more than one site. Properly controlled MHF experiments yield both the population distribution of different conformers and reactivity rate constants of the footprinting agent at all reactive positions in each conformer, which may be interpreted in terms of the accessibility of the site or the local concentration of the reagent. MHF experiments are particularly well-suited for dissecting effects at sites where unbound DNA is non-reactive and bound DNA is reactive with base-specific probes (e.g. KMnO4, DMS). We suggest that this method will also be applicable to analysis of enhancements in reactivity of other footprinting agents (e.g. DNase I, HO.). To demonstrate the utility of the MHF analysis, we quantify fragment distributions and individual site reactivities from multiple-hit KMnO4 footprinting of the non-template strand of Esigma70 RNA polymerase-lambdaPR promoter DNA complexes populated at binding equilibrium at 37 degreesC and transiently populated at a fixed time after a temperature downshift from 37 degreesC to 0 degreesC. For this system, a MHF analysis directly addresses the following questions: (i) what fraction of the population of promoter DNA molecules is open in the vicinity of the transcription start site (RPo) both at 37 degreesC and (transiently) after a downshift to 0 degreesC; (ii) does opening of the start site region in RPo occur entirely in one mechanistic step at the lambdaPR promoter and (iii) does the structure of RPo vary with temperature? In addition, we use the MHF-determined population distribution of KMnO4-reactive (RPo) and non-reactive promoter DNA to normalize the biphasic kinetics of decay of RPo to free promoter DNA after a 37 degrees to 0 degreesC temperature downshift, and thereby characterize the kinetics of the conformational changes involved in forming RPo.
J Mol Biol 1998 Nov 06
PMID:Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein-DNA complexes: application to DNA opening by Esigma70 RNA polymerase. 979 Aug 38

Base excision repair rates of dimethyl sulfate-induced 3-methyladenine and 7-methylguanine adducts were measured at nucleotide resolution along the PGK1 gene in normal human fibroblasts. Rates of 7-methylguanine repair showed a 30-fold dependence on nucleotide position, while position-dependent repair rates of 3-methyladenine varied only sixfold. Slow excision rates for 7-methylguanine bases afforded the opportunity to study their excision in vitro as a model for base excision repair. A two-component in vitro excision system, composed of human N-methylpurine-DNA glycosylase (MPG protein) and dimethyl sulfate-damaged DNA manifested sequence context-dependent rate differences for 7-methylguanine of up to 185-fold from position to position. This in vitro system reproduced both the global repair rate, and for the PGK1 coding region, the position-dependent repair patterns observed in cells. The equivalence of in vivo repair and in vitro excision data indicates that removal of 7-methylguanine by the MPG protein is the rate-limiting step in base excision repair of this lesion. DNA "repair rate footprints" associated with DNA glycosylase accessibility were observed only in a region with bound transcription factors. The "repair rate footprints" represent a rare chromatin component of 7-meG base excision repair otherwise dominated by sequence-context dependence. Comparison of in vivo repair rates to in vitro rates for 3-methyladenine, however, shows that the rate-limiting step determining position-dependent repair for this adduct is at one of the post-DNA glycosylase stages. In conclusion, this study demonstrates that a comparison of sequence context-dependent in vitro reaction rates to in vivo position-dependent repair rates permits the identification of steps responsible for position-dependent repair. Such analysis is now feasible for the different steps and adducts repaired via the base excision repair pathway.
J Mol Biol 1998 Nov 27
PMID:Heterogeneous repair of N-methylpurines at the nucleotide level in normal human cells. 981 17

The M26 meiotic recombination hot spot in the ade6 gene of Schizosaccharomyces pombe is activated by the heterodimeric M26 binding protein Mts1-Mts2. The individual Mts1 (Atf1, Gad7) and Mts2 (Pcr1) proteins are also transcription factors involved in developmental decisions. We report that the Mts proteins are key effectors of at least two distinct classes of developmental decisions regulated by the mitogen-activated protein (MAP) kinase cascade. The first class (osmoregulation, spore viability, and spore quiescence) requires the Spc1 MAP kinase and the Mts1 protein but does not require the Mts2 protein. The second class (mating, meiosis, and recombination hot spot activation) requires the Spc1 kinase and the Mts1-Mts2 heterodimer. Northern and Western blotting eliminated any significant role for the Spc1 kinase in regulating the expression levels of the Mts proteins. Gel mobility shift experiments indicated that the Mts1-Mts2 heterodimer does not need to be phosphorylated to bind to ade6-M26 DNA in vitro. However, in vivo dimethyl sulfate footprinting demonstrated that protein-DNA interaction within cells is dependent upon the Spc1 MAP kinase, which phosphorylates the Mts1 protein. Thus, the Spc1 kinase helps regulate the effector activities of the Mts1-Mts2 heterodimer in part by modulating its ability to occupy the M26 DNA site in vivo. Meiotic recombination hot spot function is likely the result of DNA conformational changes imparted by binding of the Mts1-Mts2 meiotic transcription factor.
Mol Cell Biol 1998 Dec
PMID:Regulation of the Mts1-Mts2-dependent ade6-M26 meiotic recombination hot spot and developmental decisions by the Spc1 mitogen-activated protein kinase of fission yeast. 981 43

The bacteriophage Mu mom gene encodes the unique DNA-modification function of the phage. Regulation of the mom gene at the transcriptional level is brought about by the transactivator protein C of the phage. The mom promoter is an activator-dependent weak promoter having poor -10 and -35 elements separated by a 19 bp suboptimal spacer region. These features could constrain RNA polymerase occupancy at the promoter. Here, we have probed into the mechanism by which C protein acts as a transcriptional activator at Pmom. In vivo dimethyl sulfate footprinting studies demonstrate C protein-mediated asymmetric distortion of its specific site at the mom regulatory region. Using a coupled topoisomerase assay, we demonstrate that C protein induces the unwinding of DNA. This C-mediated unwinding seems to be localised to the 3' flanking region of the C binding site located adjacent to and overlapping the -35 element of Pmom. These results suggest that C protein-mediated torsional changes could be reorienting the -10 and -35 elements to a favorable conformation for RNA polymerase occupancy at the mom promoter.
J Mol Biol 1998 Dec 11
PMID:Transcriptional activator C protein-mediated unwinding of DNA as a possible mechanism for mom gene activation. 983 13


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