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)

Antigens of Chironomus reactive with human sera containing anti-Ku antibodies and also with specific antibodies to each Ku subunit were characterized by immunoblot analysis. Three main antigen species were identified in nuclear-enriched extracts from salivary gland cells of Chironomus thummi, ranging in Mr from 55000 to 67000. The nuclear localization of Ku-related antigens in the dipteran Chironomus was studied by immunofluorescent labeling in polytene chromosomes of the salivary glands. Balbiani rings, loci highly active in transcription, were found to be strongly labeled by anti-Ku antibodies. Sugar-induced changes in the activity of the Balbiani ring genes were accompanied by the redistribution of Ku-related antigens as visualized by their absence in regressed Balbiani ring loci, and continued presence only in those that were transcriptionally active. A drastic change in the distribution of Ku-related antigens was also observed when C. thummi larvae underwent heat treatment as the immunofluorescent staining was restricted to previously described heat shock puffs. Anti-Ku sera reacted in addition with several chromosomal bands in which the presence of RNA polymerase II was also immunologically detected. The results show that Chironomus antigens reactive with anti-Ku antibodies are related to transcription in polytene chromosomes.
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PMID:Ku-related antigens are associated with transcriptionally active loci in Chironomus polytene chromosomes. 878 Nov 83

The transcription factor TFIID is a multisubunit complex that is required for promoter recognition and accurate initiation of transcription by RNA polymerase II. To dissect the molecular architecture and the biochemical properties of TFIID, a small-scale density gradient sedimentation method is employed to separate related complexes through differences in their sedimentation properties. A small amount of starting material is sufficient to obtain readily assayable amounts of separated proteins after centrifugation for 8 to 12 h in a benchtop ultracentrifuge. Gradient fractions are analyzed by immunoblotting for the presence of specific components of TFIID. Sucrose gradient sedimentation is performed to separate a mixture of multiprotein complexes from a crude nuclear extract immunoprecipitation of the proteins present in each fraction with an anti-TBP antibody reveals multiple TBP-containing complexes of different sizes. Density gradient sedimentation permits separation of specific components in a complex mixture and preserves activity, allowing functional assays.
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PMID:Small-scale density gradient sedimentation to separate and analyze multiprotein complexes. 923 67

Transcription initiation of ribosomal RNA genes requires RNA polymerase I (Pol I) and auxiliary factors which either bind directly to the rDNA promoter, e.g. TIF-IB/SL1 and UBF, or are assembled into productive transcription initiation complexes via interaction with Pol I, e.g. TIF-IA, and TIF-IC. Here we show that all components required for specific rDNA transcription initiation are capable of physical interaction with Pol I in the absence of DNA and can be co-immunoprecipitated with antibodies against defined subunits of murine Pol I. Sucrose gradient centrifugation and fractionation on gel filtration columns reveals that approximately 10% of cellular Pol I elutes as a defined complex with an apparent molecular mass of > 2000 kDa. The large Pol I complex contains saturating levels of TIF-IA, TIF-IB and UBF, but limiting amounts of TIF-IC. In support of the existence of a functional complex between Pol I and basal factors, the large complex is transcriptionally active after complementation with TIF-IC. The results suggest that, analogous to class II gene transcription, a pre-assembled complex, the "Pol I holoenzyme", exists that appears to be the initiation-competent form of Pol I.
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PMID:Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors. 945 38

Viral characterization studies were carried out on GB virus C (GBV-C) RNA positive plasma from normal human donors and from donors co-infected with GBV-C and hepatitis C virus (HCV). GBV-C RNA was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) and probe hybridization in a single tube assay. Sequential filtration of GBV-C positive plasma indicated that GBV-C RNA is associated with a particle 50-100 nm in diameter. The peak of GBV-C RNA in sucrose gradients was observed at a buoyant density of 1.05-1.13 g/ml. GBV-C RNA titer was reduced following treatment with chloroform or with five detergents indicating that GBV-C has a lipid-containing envelope. Sucrose density gradients and self-forming cesium chloride gradients of detergent-treated GBV-C showed a shift in the RNA peak to heavier buoyant density only when RNase inhibitor (RNasin) and high detergent concentrations were present. The treated material was non-filterable and the RNA had a density of > 1.5 gm/ml.
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PMID:Biophysical characterization of GB virus C from human plasma. 962 48

Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35 degreesC). In contrast, at moderate temperature (23 degreesC) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in Escherichia coli prior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.
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PMID:Rpb4, a subunit of RNA polymerase II, enables the enzyme to transcribe at temperature extremes in vitro. 982 26

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.
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PMID:Annexin A2 is a novel RNA-binding protein. 1467 33

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.
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PMID:Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells. 1818 76

Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.
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PMID:Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress. 2373 32

Commercial sugarcane (Saccharum hybrid) is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453) using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS) and 7,380 probes that hybridize to antisense transcripts (AS).We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution.
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PMID:Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop. 2393 27

The filamentous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 accumulates sucrose as a compatible solute against salt stress. Sucrose-phosphate synthase activity, which is responsible for the sucrose synthesis, is increased by salt stress, but the mechanism underlying the regulation of sucrose synthesis remains unknown. In the present study, a response regulator, OrrA, was shown to control sucrose synthesis. Expression of spsA, which encodes a sucrose-phosphate synthase, and susA and susB, which encode sucrose synthases, was induced by salt stress. In the orrA disruptant, salt induction of these genes was completely abolished. The cellular sucrose level of the orrA disruptant was reduced to 40% of that in the wild type under salt stress conditions. Moreover, overexpression of orrA resulted in enhanced expression of spsA, susA, and susB, followed by accumulation of sucrose, without the addition of NaCl. We also found that SigB2, a group 2 sigma factor of RNA polymerase, regulated the early response to salt stress under the control of OrrA. It is concluded that OrrA controls sucrose synthesis in collaboration with SigB2.
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PMID:Sucrose synthesis in the nitrogen-fixing Cyanobacterium Anabaena sp. strain PCC 7120 is controlled by the two-component response regulator OrrA. 2500 30


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