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The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.  相似文献   

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The sequence of the Kluyveromyces lactis BiP gene.   总被引:3,自引:1,他引:3       下载免费PDF全文
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Nuclear export of IkappaBalpha is mediated by the CRM1 nuclear export receptor. However, the identity of the nuclear export sequences NES(s) in IkappaBalpha that are responsible for binding of IkappaBalpha to CRM1 is controversial. Both a N-terminal NES-like region (amino acids 45-54) and a C-terminal NES-like region (amino acids 265-280) have, in a number of reports from different laboratories, been implicated in CRM1-dependent nuclear export of IkappaBalpha. We now demonstrate that the N-terminal NES-like region, but not the C-terminal NES-like region, is required for RanGTP-dependent binding of IkappaBalpha to CRM1. IkappaBalpha is a relatively weak substrate for CRM1, with an affinity for CRM1 that is 100-fold less than the minute virus of mice NS2 protein, a high affinity cargo protein for CRM1. We also demonstrate that IkappaBalpha functions as a physical adaptor between CRM1 and NFkappaB/Rel proteins. Both free IkappaBalpha and Rel-associated IkappaBalpha have comparable affinities for CRM1, suggesting that CRM1 does not discriminate between free IkappaBalpha and Rel-associated IkappaBalpha. Nuclear export of c-Rel by IkappaBalpha requires the N-terminal NES-like sequence of IkappaBalpha but is not affected by alanine substitutions within the C-terminal NES-like sequence of IkappaBalpha. In contrast, nuclear export of the v-Rel oncoprotein by IkappaBalpha is disrupted by alanine substitutions within either the N-terminal or the C-terminal NES-like sequences. However, alanine substitutions within the C-terminal NES-like sequence significantly reduce the affinity of IkappaBalpha for v-Rel, suggesting that loss of export function for this mutant is secondary to reduced association between IkappaBalpha and v-Rel. Taken together, our results demonstrate that the N-terminal NES-like sequence in IkappaBalpha is required for RanGTP-dependent binding of both free IkappaBalpha and NFkappaB/Rel-associated IkappaBalpha proteins to CRM1.  相似文献   

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During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.  相似文献   

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The Taka-amylase A gene (taaG2) of Aspergillus oryzae is inducibly expressed in A. nidulans upon exposure to inducing carbon sources, such as starch and maltose. In order to identify nuclear factor(s) possibly involved in the induction of the taaG2 gene, gel mobility shift assays and DNase I footprinting analyses were carried out, and revealed a novel nuclear factor in A. nidulans extracts, which specifically bound to two sites in the taaG2 promoter region, −204 to −189 and −182 to −168, which share the common sequence GGAAATT. The nuclear factor was detected in nuclei from both induced and uninduced mycelia. Mutational analysis within and around the binding sequences demonstrated that only the upstream binding sequence, designated SRE (starch responsive element), was required for the inducible expression of the taaG2 gene, and thus we designated the nuclear factor SREB (SRE binding factor). The downstream binding site contained an inverted SRE (ISRE) and played no role in the induction of taaG2 expression. SREB was shown by gel retardation assays to have higher affinity for SRE than for ISRE. Received: 26 January 1999 / Accepted: 10 November 1999  相似文献   

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The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.  相似文献   

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A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.  相似文献   

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Nuclear factor kappa B (NF-κB) plays a prominent role in the pathogenesis of infectious diseases. Staphylococcus aureus (S. aureus), which can attach to and invade human osteoblasts, is the most common causative agent of osteomyelitis. To determine whether S. aureus can activate NF-κB in human osteoblasts and explore the possible factors of activation in response to infection, we used flow cytometry, enzyme-linked immunosorbent assay, immunoblots, and electrophoretic mobility shift assays to quantify the invasion of bacteria, to measure the interleukin-6 (IL-6) of culture supernatants, and to investigate the IκBα degradation and NF-κB activation in human osteoblasts. Moreover, we explored the possible factors responsible for the activation of NF-κB by preventing S. aureus from physically touching human osteoblasts or inhibiting the invasion of S. aureus into human osteoblasts under co-culture conditions, by incubating proteinase K-treated or ultraviolet-killed S. aureus with human osteoblasts and by treating human osteoblasts with peptidoglycan (PGN) or lipoteichoic acid (LTA). We found that S. aureus induced the IκBα degradation and NF-κB activation, which could regulate IL-6 secretion in the culture supernatants of human osteoblasts in response to infection. In addition, the maximal IκBα degradation and NF-κB activation in human osteoblasts occurred prior to the maximal invasion of S. aureus. It was the attachment not invasion or the secreted soluble factor(s), PGN, LTA of S. aureus, that could induce the IκBα degradation and NF-κB activation in human osteoblasts. These results indicated that S. aureus can activate NF-κB in human osteoblasts and that the attachment of S. aureus is required for this activation in response to infection.  相似文献   

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