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)

Previously we have shown that nuclear extracts from mouse cells contain a heterogeneous group of polypeptides (p65, p80, p90, p100) which form distinct DNA-protein complexes on the 18 base-pair sequence element (termed Sal-box), which constitutes the murine rDNA transcription termination signal. These distinct proteins mediate cessation of RNA polymerase I (pol I) transcription elongation and release of the nascent RNA chains, indicating that they function as termination factor(s). Here, we report the biochemical analysis of the pol I-specific transcription termination factor TTFI. We show that the heterogeneity of TTFI is due to limited proteolysis of a larger, 130 kDa precursor protein (p130). The DNA-binding activity of p130 is strongly reduced as compared to the proteolytic derivatives, indicating that the DNA-binding domain is repressed within the full-length molecule. We have used limited proteolysis to purify and functionally characterize a TTFI core polypeptide (p50) which still specifically binds to the Sal-box target sequence and directs rDNA transcription termination. The equilibrium constant of purified p50 to bind specifically to DNA is 9 x 10(9) M-1. Additionally, we demonstrate that TTFI binds to DNA as a monomer and that binding induces DNA bending. This observation suggests that not only specific DNA-protein and protein-protein interactions but also conformational alterations of DNA may play a role in the termination process.
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PMID:Limited proteolysis unmasks specific DNA-binding of the murine RNA polymerase I-specific transcription termination factor TTFI. 140 80

The steady-state level of mRNA encoding the glycoprotein hormone alpha-subunit is increased about 4-fold in HeLa cells by cycloheximide (CHX) or puromycin at concentrations that inhibit protein synthesis. This effect is observed in a number of cell lines that ectopically produce alpha-subunit, including ChaGo (brochogenic carcinoma), FL (amnion), and HeLa (cervical carcinoma). No increase in alpha-subunit mRNA is evident in two choriocarcinoma cell lines (JAr, JEG-3) that produce alpha-subunit as an eutopic product. The half-life of alpha-subunit mRNA is unchanged in the presence of CHX, but nuclear run-on assays demonstrate a 2.6-fold greater loading of RNA polymerase on the alpha-subunit gene in nuclei from CHX-treated cells. These results suggest that inhibition of protein synthesis results in higher transcription rates and not in decreased mRNA turnover. A nuclear protein (Mr 50,000) that binds to a DNA fragment located 5' proximal to the alpha-subunit gene but not to more distal 5'-flanking sequence or to the alpha-subunit cDNA has been identified in HeLa but not in JEG-3 cell lines. The p50 DNA binding activity in HeLa cells decreases in the presence of CHX at a rate similar to that at which alpha-subunit mRNA increases. Moreover, in a series of HeLa cell clones, the levels of p50 are directly proportional to the magnitude of induction produced by CHX. These data are consistent with a model for alpha-subunit gene regulation involving a labile repressor and constitute yet another level of differential regulation of the alpha-subunit gene in cells that produce the hormone subunit in an ectopic versus eutopic manner.
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PMID:Induction by cycloheximide of the glycoprotein hormone alpha-subunit gene in human tumor cell lines and identification of a possible negative regulatory factor. 169 3

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by recruitment of the RNA polymerase II transcriptional machinery to the chromatin-repressed basal promoter.
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PMID:NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro. 855 93

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent activator of viral transcription. Tax also activates the expression of specific cellular genes involved in the control of T-lymphocyte growth via effects on cellular transcription factors, including members of the NF-kappaB/cRel family. Immunocytochemistry and electron microscopy were used to characterize the intracellular localization of Tax and identify cellular factors which are the potential targets for its transcriptional activity. These studies indicated that Tax localizes in discrete nuclear foci in T lymphocytes transformed by HTLV-1 and in cells transduced with Tax expression vectors. The Tax-containing foci are complex nuclear structures comprising a central core in which Tax colocalizes with splicing factor Sm. In addition to splicing factors Sm and SC-35, the Tax-containing nuclear structures also contain transcriptional components, including the largest subunit of RNA polymerase II and cyclin-dependent kinase CDK8. The inclusion of the two subunits of NF-kappaB, p50 and RelA, and the presence of the mRNA from a gene specifically activated by Tax through NF-kappaB binding sites suggest that these unique nuclear structures participate in Tax-mediated activation of gene expression via the NF-kappaB pathway.
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PMID:The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-kappaB proteins. 909 20

The transcriptional activity of an in vitro assembled human interferon-beta gene enhanceosome is highly synergistic. This synergy requires five distinct transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, and p50/p65 of NF-kappaB), the high mobility group protein HMG I(Y), and the correct alignment of protein-binding sites on the face of the DNA double helix. Here, we investigate the mechanisms of enhanceosome-dependent transcriptional synergy during preinitiation complex assembly in vitro. We show that the stereospecific assembly of the enhanceosome is critical for the efficient recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream stimulatory activity complex) and for the subsequent recruitment of the RNA polymerase II holoenzyme complex. In addition, we provide evidence that recruitment of the holoenzyme by the enhanceosome is due, at least in part, to interactions between the enhanceosome and the transcriptional coactivator CREB, cAMP responsive element binding protein (CBP). These studies reveal a unique role of enhanceosomes in the cooperative assembly of the transcription machinery on the human interferon-beta promoter.
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PMID:Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro. 977 Apr 62

Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes. Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b. Tih2p is required for vegetative cell growth and localizes in the nucleus. Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex. Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p. When a temperature-sensitive tih2 mutant, tih2-160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G(1) phase. Northern blot analysis revealed that Tih2p is required for transcription of G(1) cyclin and of several ribosomal protein genes. The similarities between the mutant phenotypes of tih2-160 and those of taf145 mutants suggest a role for TIH2 in the regulation of RNA polymerase II-directed transcription.
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PMID:The Saccharomyces cerevisiae RuvB-like protein, Tih2p, is required for cell cycle progression and RNA polymerase II-directed transcription. 1078 6

We analyzed the mechanism of human polymeric immunoglobulin receptor (pIgR) gene upregulation by tumor necrosis factor (TNF)-alpha. Northern blot analysis showed that the expression of pIgR mRNA was enhanced by TNF-alpha stimulation. This activation was completely inhibited by RNA polymerase or protein synthesis inhibitors, suggesting that the regulation of pIgR gene expression depends on de novo RNA and protein synthesis. Furthermore, the stimulation of pIgR mRNA by TNF-alpha was decreased by pyrrolidinedithiocarbamate and L-1-4'-tosylamino-phenylethyl-chloromethyl ketone, which are known nuclear factor (NF)-kappaB inhibitors. For further analysis of gene regulation, we cloned and sequenced the 1.5-kb 5'-flanking region of the pIgR gene. In the upstream region, we found two NF-kappaB-binding motifs (named kappaB1 and kappaB2 from the 5' region). An electrophoretic mobility shift assay indicated that two components of the NF-kappaB/Re1 family, p50 and p65, bound with higher affinity to the KB2 element than to the kappaB1 element. We also analyzed plgR gene expression using reporter plasmids expressing the firefly luciferase gene. Stimulation by TNF-alpha significantly activated the pIgR gene promoter, as a 775-bp upstream region of the pIgR gene increased luciferase gene expression in cells treated with TNF-alpha. The activation of promoter activity by TNF-alpha was abolished when a mutation was inserted into kappaB1 or kappaB2. These data indicated that pIgR gene expression induced by TNF-alpha is transcriptionally regulated via activation of NF-kappaB. In addition, there is a possibility that another factor may act in concert with NF-kappaB.
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PMID:Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene. 1080 41

Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor kappaB (NF-kappaB), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen alpha1[I]. In this study, we identify three NF-kappaB binding sites within the human procollagen alpha1[I] gene promoter, show that titanium particles stimulate their binding of the NF-kappaB subunits Rel A (p65) and NF-kappaB1 (p50), and find NF-kappaB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-kappaB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-kappaB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-kappaB, suggesting the involvement of redox signals in NF-kappaB-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen alpha1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen alpha1[I] mRNA. Collectively, these results show that titanium particles can activate NF-kappaB signaling in osteoblasts and suggest that NF-kappaB binding to the collagen gene promoter has a functional role in the down-regulation of procollagen alpha1[I] gene transcription.
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PMID:Down-regulation of procollagen alpha1[I]] messenger RNA by titanium particles correlates with nuclear factor kappaB (NF-kappaB) activation and increased rel A and NF-kappaB1 binding to the collagen promoter. 1127 68

Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL-6 release and suppress the gene expression of procollagens alpha1[I] and alpha1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG-63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL-8 and MCP-1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL-8 and MCP-1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL-8 and MCP-1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF-kappaB to the IL-8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL-8 and MCP-1 in human osteoblasts.
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PMID:Titanium particles induce the immediate early stress responsive chemokines IL-8 and MCP-1 in osteoblasts. 1203 22

Interleukin-6 (IL-6) secretion from endothelial cells (ECs) in response to mechanical stimuli plays an important role in the regenerative and inflammatory responses. The aim of this study was to determine the mechanism for the secretion of IL-6 from ECs in response to uni-axial continuous stretch. Continuous stretch induced IL-6 secretion from human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed that the transcription of the IL-6 gene peaked 2h after stretch. In vitro kinase assay of IkappaB kinase (IKKs) activity demonstrated that the activation of IKKs peaked 15 min after stretch. Two NF-kappaB inhibitors, pyrrolidine dithiocarbamanate (PDTC) and SN50, or antisense oligodeoxynucleotides for NF-kappaB p65 and p50 suppressed IL-6 mRNA expressions induced by continuous stretch. In conclusion, continuous stretch induces IL-6 secretion from ECs, most likely through sequential activation of IKKs and NF-kappaB.
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PMID:Stretch-induced IL-6 secretion from endothelial cells requires NF-kappaB activation. 1290 69


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