Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The region of the Xenopus laevis estrogen receptor responsible for interaction with DNA, the DNA binding domain (DBD), has been cloned and overexpressed in Escherichia coli using a T7 RNA polymerase expression system. Extracts from cells transformed with the DBD expression vector contain a single protein which reacts with polyclonal antibodies to estrogen receptor and exhibits sequence-specific binding to a DNA fragment containing a consensus estrogen response element. The DBD protein has been purified to near homogeneity. Determination of the rotational relaxation time of the dansylated DBD by fluorescence polarization and size fractionation by Superdex column chromatography indicate that the DBD is a monomer in solution. The DBD forms a single protein-estrogen response element complex in gel mobility shift assays at DBD concentrations of 18-3,600 nM, suggesting that the DBD is bound to both halves of the palindromic estrogen response element. To investigate the ability of the DBD expressed in bacteria to activate gene expression, we have developed a simple liposome-based system for delivery of protein into cultured cells. Transfected DBD protein elicited large, concentration-dependent increases in transcription of an estrogen receptor regulated reporter gene. These data demonstrate that the bacterially expressed DNA binding domain, which represents a small portion of the Xenopus laevis estrogen receptor, retains significant ability to activate transcription of an estrogen-responsive promoter in vertebrate cells.
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PMID:Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells. 174 80

The activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes. To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae. We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions. These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter. Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes. Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D. (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo.
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PMID:Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo. 191 50

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17 beta-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-I-hypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately determine the transcriptional response of the PRL gene to E2.
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PMID:An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens. 196 74

We previously reported the isolation of a partial cDNA clone encoding the rainbow trout estrogen receptor (rtER). A 0.4 kb 5'-end insert of this cDNA was used to screen the trout liver lambda gt10 cDNA library, and a full-length cDNA was isolated and sequenced. The principal structural characteristics of the complete coding sequence of the rtER are: first a remarkable homology of the DNA binding (C) and hormone binding (E) domains with those of other species, and second the lack of an A region, the function of which is not yet known but which is well conserved in other species. In vitro expression of the full-length rtER cDNA was carried out after transcription by T7 RNA polymerase and translation in rabbit reticulocyte lysate. Translation product analysis shows three major proteins, the largest one of which probably corresponds to the translation of the complete open reading frame of mRNA. The rtER in vitro translation products specifically bind estrogens (estradiol and diethylstilbestrol), without competition from testosterone or cortisol. The equilibrium dissociation constant (Kd), deduced from the Scatchard plot, is in the same order of magnitude as those determined heretofore in salmon livers during classical experiments. The tissue distribution of rtER mRNA shows that the same mRNA size (3.5 kb) is also present in the pituitary and hypothalamus. However, in the pituitary, a smaller sized mRNA (1.4 kb) is also detected.
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PMID:Full-length sequence and in vitro expression of rainbow trout estrogen receptor cDNA. 221 31

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.
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PMID:Mechanism of estrogen receptor-dependent transcription in a cell-free system. 224 75

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.
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PMID:Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand. 228 Jul 70

The estrogen receptor activation factor (E-RAF)-mediated binding of the receptor-estrogen complex to uterine nuclei was found to involve at least two classes of nuclear macromolecules: (1) the DNA, and (2) a proteinacious component. Evidences are presented to show that at least a portion of (2) is represented by the nuclear RNA polymerases. The receptor-estrogen complex associated in vivo with the nuclear RNA polymerases existed in two distinct forms which sedimented at 3.8 S and 4.8 S on sucrose density gradients. Almost 2/3 of the total radioactivity was associated with the 3.8 S species. Saturation kinetics of the two forms showed that while the 4.8 S form displayed characteristics similar to the classical type I nuclear binding site, the features displayed by the 3.8 S form were closely similar to those of the nuclear type II site. The 4.8 S species is a DNA binding form while the 3.8 S form is non-DNA binding. Anti-E-RAF IIA IgG cross-reacted with both the binding components. Goat uterine E-RAF I, IIA and IIB were purified to homogeneity as described earlier. While E-RAF IIA and IIB destabilized the native DNA structure and induced separation of the DNA strands, E-RAF I performed the opposite function. The reactions required the presence of ATP; all three of them displayed DNA-dependent ATPase activity. In an in vitro transcription system which contained purified RNA polymerase B (rat liver enzyme) and goat uterine DNA, E-RAF IIA and IIB enhanced transcription 7-fold over the control while E-RAF I totally suppressed the transcription process, irrespective of whether it was stimulated earlier by the other two E-RAF forms or not. This E-RAF property remained unchanged even after its association with the 4 S receptor-estrogen complex forming 5 S complex.
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PMID:Molecular aspects of estrogen receptor activation factor (E-RAF) function. 252 74

We transcribed a cDNA clone of the human estrogen receptor (ER) with T7 RNA polymerase. The 32P-cRNA transcript complementary to ER mRNA was hybridized to poly(A)+ RNA from human uterus and revealed a single band of approximately 4.2 kilobases. No hybridization was seen with the cRNA probe of the opposite orientation. Hybridization of total RNA from calf and rat uterus yielded a single band at approximately 3.8 kilobases for both species. Total RNA from rat spleen did not hybridize. A 35S-labeled cRNA probe was prepared for in situ hybridization of ER mRNA in human uterus and spleen. Autoradiographic signal was present over endometrial epithelium, stromal cells of the lamina propria, and smooth muscle cells of the myometrium but was absent from sections of spleen. The ER mRNA hybridization label was located over cytoplasm and nuclei of uterine target cells.
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PMID:Detection of estrogen receptor mRNA in human uterus. 365 5

Template-engaged and total RNA polymerase II molecules were quantitated in isolated nuclei at various stages of estrogen withdrawal and secondary stimulation by using [3H]amanitin titration assays. Estrogen receptors, RNA transcriptional activity, and ovalbumin mRNA were also measured, and comparisons were made between these parameters to determine whether any significant correlations exist. In isolated nuclei, the highest positive correlations existed between template-engaged RNA polymerase II, ovalbumin mRNA synthesis in vitro, and estrogen receptor concentration. Interestingly, restimulation of estrogen-withdrawn chicks results in replenishment of RNA polymerase II activity to prewithdrawal levels within 4 h; however, the recovery of the numbers of template-engaged polymerase II molecules, ovalbumin gene transcription, and nuclear receptor binding lags behind. These findings suggest that the estrogen effect on RNA polymerase activity is more rapid than the increase in template-engaged RNA polymerase II and ovalbumin-specific gene transcription. The excellent correlation that exists between nuclear estrogen receptor concentrations, template-engaged RNA polymerase II, and ovalbumin gene transcription strongly supports the hypothesis that estrogen receptors mediate RNA polymerase II binding to sequences associated with preferential transcription of the ovalbumin gene.
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PMID:Correlation in isolated nuclei of template-engaged RNA polymerase II, ovalbumin mRNA synthesis, and estrogen receptor concentrations. 398 75

Preliminary evidence has indicated that the number of nuclear bodies in uterine luminal epithelial cells of the immature rat may be related to the duration of nuclear retention of the estrogen receptor complex (Clark et al., 1978). To test this hypothesis, an ultrastructural analysis of nuclear and cytoplasmic differentiation was performed at 4, 12, 24, 48, and 72 hr after a single injection of estradiol or nafoxidine (synthetic estrogen agonist/antagonist) into 21 day female rats. Variations in nuclear and cytoplasmic differentiation and in the frequency of occurrence of nuclear bodies (simple and complex) were determined and compared with established biochemical changes in the concentration of nuclear estrogen receptor and RNA polymerase activity (Clark et al., 1978). Following nafoxidine there is sustained elevation of the nuclear concentration of the estrogen receptor as well as RNA polymerase I and II activities over the entire 72-hr period. From 4 to 72 hr the height of the luminal epithelial cell as well as the frequency of nuclear bodies increase at linear rates. Through steady expansion of the cytoplasmic membrane system (RER) and Golgi) the relatively undifferentiated epithelial cells of the control uterus are converted progressively into ones equipped for protein secretion. At 72 hr the effects of an estradiol implant resemble closely those observed after a single injection of nafoxidine; these include sustained nuclear receptor occupancy, elevated RNA polymerase activity, epithelial hypertrophy, and high frequency of nuclear bodies. However, after a single injection of estradiol, the luminal epithelial cells become slightly but significantly taller than the control cells and remain close to this size from 24 to 72 hr.; the frequency of nuclear bodies decreases linearly from 4 to 72 hr to fall below the control level. In addition, limited cytoplasmic autolysis is evident from 24 to 72 hr. A single injection of estradiol results in short-term nuclear receptor occupancy and elevated RNA polymerase activities which return to control levels by 24 hr. This collective evidence offers further support to the hypothesis that the duration of nuclear occupancy by the estrogen receptor is reflected in the size of the nuclear body populations in these epithelial target cells. Also during hyperestrogenization, epithelial hypertrophy is accompanied by steady formation of nuclear bodies.
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PMID:Nuclear bodies as structural indicators of estrogenic stimulation in uterine luminal epithelial cells. 734 May 72


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