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Transcription Activation (transcription + activation)
Selected AbstractsA novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutrophaMOLECULAR MICROBIOLOGY, Issue 3 2000Anne Pohlmann Ralstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N2O) with two isofunctional single component membrane-bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co-transcribed with norA1 from a ,54 (RpoN)-dependent promoter, located upstream of norA1. With the aid of norA1,,lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase-deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO-donating agent sodium nitroprusside caused induction of norA1,,lacZ transcription. Truncation of the N-terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1,,lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co-ordinated on the regulatory level with the other nitrogen oxide-reducing steps of the denitrification chain that are independent of NorR. [source] Steroid and thyroid hormone receptors in mitochondriaIUBMB LIFE, Issue 4 2008Anna-Maria G. Psarra Abstract Receptors for glucocorticoids, estrogens, androgens, and thyroid hormones have been detected in mitochondria of various cell types by Western blotting, immunofluorescence labeling, confocal microscopy, and immunogold electron microscopy. A role of these receptors in mitochondrial transcription, OXPHOS biosynthesis, and apoptosis is now being revealed. Steroid and thyroid hormones regulate energy production, inducing nuclear and mitochondrial OXPHOS genes by way of cognate receptors. In addition to the action of the nuclearly localized receptors on nuclear OXPHOS gene transcription, a parallel direct action of the mitochondrially localized receptors on mitochondrial transcription has been demonstrated. The coordination of transcription activation in nuclei and mitochondria by the respective receptors is in part realized by their binding to common trans acting elements in the two genomes. Recent evidence points to a role of the mitochondrial receptors in cell survival and apoptosis, exerted by genomic and nongenomic mechanisms. The identification of additional receptors of the superfamily of nuclear receptors and of other nuclear transcription factors in mitochondria increases their arsenal of regulatory molecules and further underlines the central role of these organelles in the integration of growth, metabolic, and cell survival signals. © 2008 IUBMB IUBMB Life, 60(4): 210,223, 2008 [source] The DEAD-box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB-mediated transcriptionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Musarat Ishaq Abstract DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-,B), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-,B dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-,B-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-,B-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-,B-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-,B transcriptional activity. J. Cell. Biochem. 106: 296,305, 2009. © 2008 Wiley-Liss, Inc. [source] H4 acetylation does not replace H3 acetylation in chromatin remodelling and transcription activation of Adr1-dependent genesMOLECULAR MICROBIOLOGY, Issue 5 2006Eleonora Agricola Summary Histone acetylation regulates gene expression. Whether this is caused by a general increase in nucleosome fluidity due to charge neutralization or by a more specific code is still matter of debate. By using a set of glucose-repressed Adr1-dependent genes of Saccharomyces cerevisiae, whose transcription was previously shown to require both Gcn5 and Esa1, we asked how changes of histone acetylation patterns at the promoter nucleosomes regulate chromatin remodelling and activation. When the signal of glucose reduction reaches the cells, H4 acetylation is kept constant while an increase of H3 acetylation occurs, in an Adr1- and Gcn5-dependent manner. In cells lacking Gcn5 activity, the H3 acetylation increase does not occur and an unexpected increase of histone H4 acetylation is observed. Nevertheless, chromatin remodelling and transcription activation are impaired, suggesting that acetylation of H3 and H4 histones plays different roles. [source] Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulonMOLECULAR MICROBIOLOGY, Issue 4 2005Kevin L. Griffith Summary In Escherichia coli, SoxS, MarA and Rob form a closely related subset of the AraC/XylS family of positive regulators, sharing ,42% amino acid sequence identity over the length of SoxS and the ability to activate transcription of a common set of target genes that provide resistance to redox-cycling compounds and antibiotics. On the basis of its ,43% amino acid sequence identity with SoxS, MarA and Rob, TetD, encoded by transposon Tn10, appears to be a fourth member of the subset. However, although its expression has been shown to be negatively regulated by TetC and not inducible by tetracycline, the physiological function of TetD is unknown. Accordingly, in the work presented here, we initiate a molecular characterization of TetD. We show that expression of TetD activates transcription of a subset of the SoxS/MarA/Rob regulon genes and confers resistance to redox-cycling compounds and antibiotics. We show that mutations in the putative TetD binding site of a TetD-activatable promoter and a mutation in the protein's N-terminal DNA recognition helix interfere with transcription activation, thereby indicating that TetD directly activates target gene transcription. Finally, we show that TetD, like SoxS and MarA, is intrinsically unstable; however, unlike SoxS and MarA, TetD is not degraded by Lon or any of the cell's known cytoplasmic ATP-dependent proteases. Thus, we conclude that TetD is a bona fide member of the SoxS/MarA/Rob subfamily of positive regulators. [source] Communication between E,54, promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF ,54 -dependent activator during transcription activationMOLECULAR MICROBIOLOGY, Issue 2 2004Patricia Bordes Summary Conversion of E,54 closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of E,54 activators is used in an energy coupling reaction to remodel the E,54 closed promoter complex and to overcome the ,54 -imposed block on open complex formation. The remodelling target for the AAA activator within the E,54 closed complex includes a complex interface contributed to by Region I of ,54, core RNA polymerase and a promoter DNA fork junction structure, comprising the E,54 regulatory centre. One ,54 binding surface on E,54 activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of ,54 and the Escherichia coli AAA ,54 activator Phage shock protein F. Using E,54 promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the E,54 regulatory centre, and in particular the conformation adopted by the ,54 Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif. [source] Expression of intracellular calcium signalling genes in cattle skin during tick infestationPARASITE IMMUNOLOGY, Issue 4 2009N. BAGNALL SUMMARY It is widely acknowledged that changes in intracellular calcium ion (Ca2+) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real-time PCR was used to analyse gene expression of 14 Ca2+ signalling proteins in skin obtained from high tick-resistant (HR) and low tick-resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up-regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca2+ signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca2+ and its associated proteins might play an important role in host response to ticks and that further investigation is warranted. [source] Structure of the Escherichia coli RNA polymerase , subunit C-terminal domainACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Samuel Lara-González The , subunit C-terminal domain (,CTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli,CTD (, subunit residues 245,329) determined to 2.0,Ĺ resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P21 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, Rfree = 0.236) has improved geometry compared with prior lower resolution determinations of the ,CTD structure [Jeon et al. (1995), Science, 270, 1495,1497; Benoff et al. (2002), Science, 297, 1562,1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of ,CTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction. [source] Pathogenic T cells in murine lupus exhibit spontaneous signaling activity through phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathwaysARTHRITIS & RHEUMATISM, Issue 4 2003Florin Niculescu Objective To determine the activation status of two cytoplasmic signaling pathways, phosphatidylinositol 3-kinase (PI 3-kinase) and the mitogen-activated protein kinase (MAPK) family. Methods We studied the pathogenic CD4+ T cells that drive disease in the parent-into-F1 mouse model of lupus-like chronic graft-versus-host disease (GVHD). We determined immunoprecipitated kinase activity for PI 3-kinase and MAPK members (Raf-1, extracellular signal,regulated kinase 1 [ERK-1], c-Jun N-terminal kinase 1 [JNK-1], and p38 MAPK) from either unfractionated splenocytes or purified donor CD4+ T cells. Uninjected normal mice served as negative controls, and acute GVHD mice served as positive controls. Results Compared with negative controls, unfractionated splenocyte kinase activity from chronic GVHD mice was significantly increased for PI 3-kinase and JNK-1, but not for Raf-1, p38 MAPK, or ERK-1. Increased PI 3-kinase and JNK-1 activity was also seen in acute GVHD splenocytes, as was increased Raf-1 and p38 MAPK activity. The pattern of increased PI 3-kinase and JNK-1 activity seen in unfractionated chronic GVHD splenocytes was also seen in isolated donor, but not host, CD4+ T cells from chronic GVHD mice, indicating that donor CD4+ T cell signaling activity accounted for at least a portion of the activity observed in unfractionated splenocytes. Increased ERK-1 activity was not seen in either donor or host CD4+ T cells. This pattern of cytoplasmic signaling pathway in donor CD4+ T cells was associated with increased T cell receptor membrane signaling activation (Lck and Fyn phosphorylation) and increased transcription activation (phosphorylation of inhibitor of nuclear factor ,B), confirming the biologic significance of these observations. Conclusion The pathogenic T cells driving disease in this murine model exhibit activation in the form of spontaneous cytoplasmic signaling pathway activity that can be detected without in vitro restimulation and involves a T cell,specific (PI 3-kinase) and a nonspecific stress/cytokine pathway (JNK-1). These results raise the possibility that a full characterization of the signaling pathways active in pathogenic lupus T cells might lead to new therapeutic targets. [source] Transcription factor Sp1 regulates expression of cancer-associated molecule CD147 in human lung cancerCANCER SCIENCE, Issue 6 2010Ling-Min Kong CD147 is a novel cancer-associated biomarker that plays an important role in the invasion and metastasis of human lung cancer. In spite of its many known functions, little is known about CD147 transcriptional regulation. In this study, we explored the regulation of CD147 in human lung cancer tissues. Over 60% of the human lung cancer tissues expressed differential high levels of CD147. We then cloned the 5,-flanking region of the human CD147 gene and identified a critical promoter region at ,108 to ,42 which contained one binding site for Sp1, which was essential in up-regulating CD147 promoter activity. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and up-regulating Sp1 using transfection with eukaryotic expression vector. Consistent with the CD147 transcription activation, a high level of Sp1 expression was detected in lung cancer cell lines overexpressing CD147. Chromatin immunoprecipitation assay showed that much more Sp1 could bind to the CD147 promoter in 95-D with CD147 high expression than in SK-MES-1 with CD147 low expression. There was a significant positive correlation between CD147 expression and Sp1 expression level detected by immunohistochemistry (r = 0.831). Collectively, our results suggest that Sp1 is essential for regulating the CD147 gene expression in human lung cancer. (Cancer Sci 2010) [source] Effects of PKC , on early genome transcription activation in mouse 1-cell stage fertilized eggsCELL BIOCHEMISTRY AND FUNCTION, Issue 6 2007Bing-zhi Yu Abstract Effects of PKC , on the activation of embryonic transcription in 1-cell stage fertilized mouse eggs were explored. The effects of PKC antagonist calphostin C and PKC , specific inhibitor on the activation of embryonic early transcription were observed by Western blotting and cell immunofluorescence. PKC activity increased gradually from G1 phase to late G2 phase in mouse 1-cell stage fertilized eggs, and reached a maximum in G2 stage. Calphostin C inhibited PKC activity by about 47% in 1-cell stage fertilized eggs. Calphostin C inhibited early transcription in 1-cell stage fertilized eggs (p,<,0.01). PKC ,-Thr410 in G2 were about 27% and 110% higher than those in G1 phase of 1-cell stage fertilized eggs and MII oocytes, respectively. PKC , specific inhibitor can also inhibit early transcription in 1-cell stage fertilized eggs (p,<,0.05). The results suggest that PKC , participates in early transcription activation in mouse 1-cell stage fertilized eggs. Copyright © 2006 John Wiley & Sons, Ltd. [source] Inhibition of cyclin-dependent kinases by olomoucine and roscovitine reduces lipopolysaccharide-induced inflammatory responses via down-regulation of nuclear factor ,BCELL PROLIFERATION, Issue 2 2009R-S. Jhou Objectives:, Initiation and maintenance of pro-inflammatory reactions elicited by bacterial lipopolysaccharide and/or cytokines in the macrophage lineage have been reported to play a crucial role in acute and chronic pathogenic effects. Whether pro-inflammatory responses triggered by lipopolysaccharide in growth arrested cells differ from those in proliferating cells remains unanswered. Materials and methods:, Olomoucine and roscovitine are cyclin-dependent kinase (CDK) inhibitors that prevent progression through the cell cycle. After treatment with CDK inhibitors, expression of pro-inflammatory genes was analysed by reverse transcriptase,polymerase chain reaction. Protein levels of inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-,B) were determined by Western blotting. Promoter activity of iNOS was measured by the luciferase activity assay. Results:, In this study we have demonstrated that both olomoucine and roscovitine inhibit cell proliferation and diminish nitric oxide production and cytokine gene expression, in lipopolysaccharide-stimulated murine RAW264.7 macrophages. In addition, olomoucine reduces iNOS promoter activity and alleviates NF-,B transcription activation. After co-transfection with E2F1 interference RNA, suppression of lipopolysaccharide-mediated iNOS promoter activity and NF-,B activation was observed. Furthermore, we demonstrated that olomoucine-induced growth arrested cells reduce expression of the p65 subunit of NF-,B. Conclusions:, The findings of this study suggest that inhibition of cell-cycle progression is capable of reducing pro-inflammatory responses via down-regulation of NF-,B. 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