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)

TAP tags and dot blot analysis have been used to measure the amounts of RNA polymerase II transcription proteins in crude yeast extracts. The measurements showed comparable amounts of RNA polymerase II, TFIIE, and TFIIF, lower levels of TBP and TFIIB, and still lower levels of Mediator and TFIIH. These findings are consistent with the presumed roles of the transcription proteins, but do not support the idea of their recruitment in a single large complex to RNA polymerase II promoters. The approach employed here can be readily extended to quantitative analysis of the entire yeast proteome.
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PMID:Quantitation of the RNA polymerase II transcription machinery in yeast. 1159 27

Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon-exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense-mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap-binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap-binding protein eIF4E. These and other data indicate that NMD targets CBP80-bound mRNA during a 'pioneer' round of translation, but whether nuclear eIF4E also binds nascent but dead-end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron-containing RNA and the C-terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80-bound but not eIF4E-bound mRNA. Each of these proteins is also detected on CBP80-bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.
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PMID:The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling. 1209 54

Under visible irradiation, [Ru(TAP)2(phen)]2+(Cl-)2, [Ru(TAP)2(POQ-Nmet)]2+(Cl-)2 and [Ru(bpy)2(phen)]2+(Cl-)2 were able to dramatically reduce the in vitro transcription of a plasmid DNA template by a bacteriophage RNA polymerase. This photoactivity is related to two different mechanisms of reactivity towards DNA exhibited by these complexes under illumination.
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PMID:In vitro inhibition of gene transcription by novel photo-activated polyazaaromatic ruthenium(II) complexes. 1212 77

The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin-related system (URM1-UBA4 ) and KTI11 enhances post-translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co-fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator-nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS- and karyopherin-dependent nuclear import.
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PMID:Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification. 1294 Sep 88

The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties.
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PMID:Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer. 1751 66

The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.
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PMID:Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription. 1784 38

Downregulation of the transporter associated with antigen processing 1 (TAP-1) has been observed in many tumors and is closely associated with tumor immunoevasion mechanisms, growth, and metastatic ability. The molecular mechanisms underlying the relatively low level of transcription of the tap-1 gene in cancer cells are largely unexplained. In this study, we tested the hypothesis that epigenetic regulation plays a fundamental role in controlling tumor antigen processing and immune escape mechanisms. We found that the lack of TAP-1 transcription in TAP-deficient cells correlated with low levels of recruitment of the histone acetyltransferase, CBP, to the TAP-1 promoter. This results in lower levels of histone H3 acetylation at the TAP-1 promoter, leading to a decrease in accessibility of the RNA polymerase II complex to the TAP-1 promoter. These observations suggest that CBP-mediated histone H3 acetylation normally relaxes the chromatin structure around the TAP-1 promoter region, allowing transcription. In addition, we found a hitherto-unknown mechanism wherein interferon gamma up-regulates TAP-1 expression by increasing histone H3 acetylation at the TAP-1 promoter locus. These findings lie at the heart of understanding immune escape mechanisms in tumors and suggest that the reversal of epigenetic codes may provide novel immunotherapeutic paradigms for intervention in cancer.
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PMID:Epigenetic control of the immune escape mechanisms in malignant carcinomas. 1787 43

The mechanisms of influenza A virus mRNA intracellular transport are still not clearly understood. Here, we visualized the distribution and transport of influenza A virus mRNA in living cells using molecular beacon (MB) technology. Confocal-FRAP measurements determined that the transport of influenza A virus intronless mRNA, in both nucleus and cytoplasm, was energy dependent, being similar to that of Poly(A)(+) RNA. Drug inhibition studies in living cells revealed that the export of influenza A virus mRNA is independent of the CRM1 pathway, while the function of RNA polymerase II (RNAP-II) may be needed. In addition, viral NS1 protein and cellular TAP protein were found associated with influenza A virus mRNA in the cell nucleus. These findings characterize influenza A virus mRNA transport in living cells and suggest that influenza A virus mRNA may be exported from the nucleus by the cellular TAP/p15 pathway with NS1 protein and RNAP-II participation.
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PMID:Imaging and characterizing influenza A virus mRNA transport in living cells. 1865 28

Herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a multifunction functional protein that interacts with many cellular proteins. A number of the proteins with which ICP27 interacts require that both the N and C termini of ICP27 are intact. These include RNA polymerase II, TAP/NXF1, and Hsc70. We tested the possibility that the N and C termini of ICP27 could undergo a head-to-tail intramolecular interaction that exists in open and closed configurations for different binding partners. Here, we show by bimolecular fluorescence complementation (BiFC) assays and fluorescence resonance energy transfer (FRET) by acceptor photobleaching that ICP27 undergoes a head-to-tail intramolecular interaction but not head-to-tail or tail-to-tail intermolecular interactions. Substitution mutations in the N or C termini showed that the leucine-rich region (LRR) in the N terminus and the zinc finger-like region in the C terminus must be intact for intramolecular interactions. A recombinant virus, vNC-Venus-ICP27, was constructed, and this virus was severely impaired for virus replication. The expression of NC-Venus-ICP27 protein was delayed compared to ICP27 expression in wild-type HSV-1 infection, but NC-Venus-ICP27 was abundantly expressed at late times of infection. Because the renaturation of the Venus fluorescent protein results in a covalent bonding of the two halves of the Venus molecule, the head-to-tail interaction of NC-Venus-ICP27 locks ICP27 in a closed configuration. We suggest that the population of locked ICP27 molecules is not able to undergo further protein-protein interactions.
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PMID:Herpes simplex virus 1 regulatory protein ICP27 undergoes a head-to-tail intramolecular interaction. 2016 36

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.
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PMID:Highly reproducible label free quantitative proteomic analysis of RNA polymerase complexes. 2104 97


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