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 RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.
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PMID:The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein. 863 57

The human RNA polymerase II and III snRNA promoters share a common basal element, the proximal sequence element (PSE), which is recognized by a complex we refer to as the snRNA-activating protein complex (SNAPc). Biochemical purifications suggest that SNAPc is composed of at least four polypeptides of 43, 45, 50 and 190 kDa, as well as variable amounts of the TATA box binding protein, TBP. cDNAs encoding the 43 and 45 kDa subunits, SNAP43 and SNAP45, have been isolated, but there is no evidence that either of these subunits contacts DNA. Here we report the isolation of cDNAs encoding the 50 kDa subunit of SNAPc, SNAP50. The open reading frame predicts a 411 amino acid protein, which contains two potential zinc finger motifs. Depletions with anti-SNAP50 antibodies inhibit RNA polymerase II and III snRNA gene transcription in vitro. SNAP50 interacts with SNAP43 in co-immunoprecipitation experiments, but not with SNAP45 or TBP. UV cross-linking experiments suggest that SNAP50 contacts DNA in the SNAP complex. These results are consistent with the same core SNAP complex recognizing the PSEs of RNA polymerase II and III snRNA promoters, and provide an initial view of the architecture of the SNAP complex.
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PMID:Cloning and characterization of SNAP50, a subunit of the snRNA-activating protein complex SNAPc. 900 88

The human RNA polymerase II and III snRNA promoters have similar enhancers, the distal sequence elements (DSEs), and similar basal promoter elements, the proximal sequence elements (PSEs). The DSE, which contains an octamer motif, binds broadly expressed activator Oct-1. The PSE binds a multiprotein complex referred to as SNAPc or PTF. On DNAs containing both an octamer site and a PSE, Oct-1 and SNAPc bind cooperatively. SNAPc consists of at least four stably associated subunits, SNAP43, SNAP45, SNAP50, and SNAP190. None of the three small subunits, which have all been cloned, can bind to the PSE on their own. Here we report the isolation of cDNAs corresponding to the largest subunit of SNAPc, SNAP190. SNAP190 contains an unusual Myb DNA binding domain consisting of four complete repeats (Ra to Rd) and a half repeat (Rh). A truncated protein consisting of the last two SNAP190 Myb repeats, Rc and Rd, can bind to the PSE, suggesting that the SNAP190 Myb domain contributes to recognition of the PSE by the SNAP complex. SNAP190 is required for snRNA gene transcription by both RNA polymerases II and III and interacts with SNAP45. In addition, SNAP190 interacts with Oct-1. Together, these results suggest that the largest subunit of the SNAP complex is involved in direct recognition of the PSE and is a target for the Oct-1 activator. They also provide an example of a basal transcription factor containing a Myb DNA binding domain.
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PMID:The large subunit of basal transcription factor SNAPc is a Myb domain protein that interacts with Oct-1. 941 84

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised 'neuronal' serotonin transporter.
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PMID:Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles. 969 37

The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. alpha-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. An in vitro binding study showed that both wild-type alpha-SNAP and C-terminal-deleted alpha-SNAP mutant (1-285) can bind to syntaxin 1A. alpha-SNAP mutant (1-285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of alpha-SNAP mutant (1-285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous alpha-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if alpha-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic beta cells, the functional role of alpha-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of alpha-SNAP mutant (1-285) overexpression on GABA release. We conclude that 1) alpha-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic beta cells; and 2) the interaction of alpha-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.
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PMID:alpha-soluble N-ethylmaleimide-sensitive factor attachment protein is expressed in pancreatic beta cells and functions in insulin but not gamma-aminobutyric acid secretion. 1007 5

Transforming growth factor (TGF)-beta1 is a growth factor involved in the mechanisms of lung repair and fibrosis that follow inflammatory processes. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by inflammatory cells and the expression of TGF-beta1 by alveolar epithelial cells. Exposure of the A549 lung epithelial cell line to either an ROI generating system (xanthine and xanthine oxidase) or an RNI donor (S-nitroso-N-acetyl-penicillamine [SNAP]) promoted a time- and dose-dependent increase in TGF-beta1 release, as measured by a specific enzyme-linked immunosorbent assay. At the peak, the levels of TGF-beta1 were twice the control values. The induction of TGF-beta1 release by ROI was blunted by catalase and unaffected by superoxide dismutase, indicating the involvement of hydrogen peroxide. The response was also blunted by 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific RNA polymerase II inhibitor, and accompanied by a corresponding increase in TGF-beta1 messenger RNA, as measured by quantitative/competitive reverse transcription polymerase chain reaction, suggesting the involvement of transcriptional mechanisms and possibly other downstream mechanisms. In contrast, RNI-induced TGF-beta1 release was unaffected by DRB and blunted by the protein synthesis inhibitor cycloheximide, suggesting the involvement of translational and post-translational mechanisms. This response required cyclic guanosine monophosphate (cGMP)- mediated processes because (1) immunoreactive cGMP accumulated in the culture medium of SNAP-treated cells; (2) SNAP-induced TGF-beta1 release was blunted by KT 5823, an inhibitor of cGMP-dependent protein kinase; and (3) similar increase in TGF-beta1 release was obtained by cell exposure to membrane-permeable dibutyryl-cGMP or to atrial natriuretic factor, a known agonist of particulate guanylate cyclase. These data suggest that in vitro exposure of human alveolar epithelial cells to ROI and RNI enhances TGF-beta1 release through different mechanisms. In vivo, this control may constitute a molecular link between inflammatory and fibrotic processes.
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PMID:Reactive oxygen and nitrogen intermediates increase transforming growth factor-beta1 release from human epithelial alveolar cells through two different mechanisms. 1038 1

snRNA gene transcription is activated in part by recruitment of SNAP(c) to the core promoter through protein-protein contacts with the POU domain of the enhancer-binding factor Oct-1. We show that a mini-SNAP(c) consisting of a subset of SNAP(c) subunits is capable of directing both RNA polymerase II (Pol II) and Pol III snRNA gene transcription. Mini-SNAP(c) cannot be recruited by Oct-1, but binds as efficiently to the promoter as SNAP(c) together with Oct-1 and directs activated RNA Pol III transcription. Thus, SNAP(c) represses its own binding to DNA, and repression is relieved by interactions with the Oct-1 POU domain that promote cooperative binding. We have shown previously that TBP also represses its own binding, and in that case repression is relieved by cooperative interactions with SNAP(c). This may represent a general mechanism to ensure that core promoter-binding factors, which have strikingly slow off-rates, are recruited specifically to promoter sequences rather than to cryptic-binding sites in the genome.
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PMID:SNAP(c): a core promoter factor with a built-in DNA-binding damper that is deactivated by the Oct-1 POU domain. 1042 33

The nucleation of RNA polymerases I-III transcription complexes is usually directed by distinct multisubunit factors. In the case of the human RNA polymerase II and III small nuclear RNA (snRNA) genes, whose core promoters consist of a proximal sequence element (PSE) and a PSE combined with a TATA box, respectively, the same multisubunit complex is involved in the establishment of RNA polymerase II and III initiation complexes. This factor, the snRNA-activating protein complex or SNAP(c), binds to the PSE of both types of promoters and contains five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. SNAP(c) binds cooperatively with both Oct-1, an activator of snRNA promoters, and in the RNA polymerase III snRNA promoters, with TATA-binding protein, which binds to the TATA box located downstream of the PSE. Here we have defined subunit domains required for SNAP(c) subunit-subunit association, and we show that complexes containing little more than the domains mapped here as required for subunit-subunit contacts bind specifically to the PSE. These data provide a detailed map of the subunit-subunit interactions within a multifunctional basal transcription complex.
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PMID:A map of protein-protein contacts within the small nuclear RNA-activating protein complex SNAPc. 1105 76

The retinoblastoma protein (RB) represses RNA polymerase III transcription effectively both in vivo and in vitro. Here we demonstrate that the general transcription factors snRNA-activating protein complex (SNAP(c)) and TATA binding protein (TBP) are important for RB repression of human U6 snRNA gene transcription by RNA polymerase III. RB is associated with SNAP(c) as detected by both coimmunoprecipitation of endogenous RB with SNAP(c) and cofractionation of RB and SNAP(c) during chromatographic purification. RB also interacts with two SNAP(c) subunits, SNAP43 and SNAP50. TBP or a combination of TBP and SNAP(c) restores efficient U6 transcription from RB-treated extracts, indicating that TBP is also involved in RB regulation. In contrast, the TBP-containing complex TFIIIB restores adenovirus VAI but not human U6 transcription in RB-treated extracts, suggesting that TFIIIB is important for RB regulation of tRNA-like genes. These results suggest that different classes of RNA polymerase III-transcribed genes have distinct general transcription factor requirements for repression by RB.
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PMID:The retinoblastoma tumor suppressor protein targets distinct general transcription factors to regulate RNA polymerase III gene expression. 1109 70

The human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAP(c), and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAP(c) and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB", a human homologue of the B" subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAP(c), recombinant TBP, recombinant hB", and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components.
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PMID:Reconstitution of transcription from the human U6 small nuclear RNA promoter with eight recombinant polypeptides and a partially purified RNA polymerase III complex. 1127 1


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