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

Biological properties of an AIDS agent first isolated from a native citizen in the USSR are presented. The source of the virus was a young Byelorussian woman who in the near past had had sexual contacts with a citizen from one of the Central Africa countries. The isolate is thought to be of HIV-I type. It replicated perfectly in many continuous lymphocyte lines and had HIV-characteristic morphology. The protein spectrum of the isolate was gp120, gp41, p65/51, p55, p32, p24, p17. Reverse transcriptase activity was detected in the culture fluid of the virus-containing cell cultures. The isolate was designated HIV-IZ.
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PMID:[The biological properties of the HIV isolated from a virus carrier living in the Byelorussian SSR]. 214 58

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 RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.
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PMID:Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM. 889 45

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

The RNA polymerase gene of the murine coronavirus mouse hepatitis virus (MHV) encodes a polyprotein of greater than 750 kDa. The amino-terminal cleavage product of the MHV polymerase polyprotein, p28, has been shown to be cleaved from the polyprotein by the virus-encoded protease PCP-1. We aim to identify the MHV-JHM proteolytic products downstream of p28 and to determine which viral proteinase domains are responsible for generating each of them. To this end, we have generated antisera directed at specific MHV-JHM ORF1a regions and have used these antisera to identify six viral proteins, representing a large portion of ORF1a, from MHV-JHM-infected cells. These proteins include p28, p72, p65, p250, p210, and p27.
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PMID:Maturation of the polymerase polyprotein of the coronavirus MHV strain JHM involves a cascade of proteolytic processing events. 978 75

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

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.
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PMID:Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression. 1083 65

To investigate the role of chromatin structure in the regulation of transcription by RNA polymerase II, we developed a chromatin transcription system in which periodic nucleosome arrays are assembled with purified recombinant ATP-utilizing chromatin assembly and remodeling factor (ACF), purified recombinant nucleosome assembly protein 1 (dNAP1), purified native core histones, plasmid DNA, and ATP. With this chromatin, we observed robust activation of transcription with three different transcription factor sets (nuclear factor kappaB p65 + Sp1, estrogen receptor, and Gal4-VP16) added either before or after chromatin assembly. In fact, the efficiency of activated transcription from the ACF + dNAP1-assembled chromatin was observed to be comparable with that from naked DNA templates or chromatin assembled with a crude Drosophila extract (S190). With ACF + dNAP1-assembled chromatin, we found that transcriptional activation is dependent upon acetyl-CoA. This effect was not seen with naked DNA templates or with crude S190-assembled chromatin. We further determined that acetyl-CoA is required at the time of preinitiation complex assembly but not during assembly of the chromatin template. These findings suggest that there is at least one key acetylation event that is needed to assemble a functional transcription preinitiation complex with a chromatin template.
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PMID:Transcriptional analysis of chromatin assembled with purified ACF and dNAP1 reveals that acetyl-CoA is required for preinitiation complex assembly. 1105 7

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


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