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)

GreA is a 17.6 kDa protein from Escherichia coli that induces cleavage of the nascent transcript in the elongating complex of RNA polymerase, followed by release of the 3'-terminal fragment. Crystals of GreA have been obtained from polyethylene glycol 4000, 2-propanol and sodium citrate, pH 5.6 and have been propagated by a novel seeding procedure. The crystals diffract beyond 2 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 101.7 A, b = 42.22 A, c = 40.05 A and with one molecule in the asymmetric unit.
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PMID:Crystallization of GreA, a transcript cleavage factor from Escherichia coli. 793 13

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.
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PMID:Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells. 986 47

A Bacillus subtilis veg mutant exhibited a small reduction of absorbance, a large reduction of hexosamine release, and slow dipicolinic acid release from spores during germination with L-alanine as a germinant. But veg spores exhibited normal resistance to chloroform, 2-propanol, lysozyme, and heat. SDS-polyacrylamide gel electrophoresis of spore coat proteins revealed no difference in coat proteins between the wild type and the veg mutant. Northern and veg-lacZ fusion analyses indicated that the veg gene is transcribed in both the vegetative growth and sporulation phases, and primer extension analysis indicated an identical transcriptional start point in both phases. The upstream sequence suggests that veg is transcribed by Esigma(A) RNA polymerase. Veg-GFP fusion protein was detected for vegetative cells and spores, but the fluorescence of mother cells disappeared completely in the late sporulation phase. Decoated spores containing Veg-GFP exhibited a strong green fluorescence in the core, but much weaker fluorescence in the cortex.
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PMID:Transcriptional, functional and cytochemical analyses of the veg gene in Bacillus subtilis. 1276 Dec 95

The diversity of Paenibacillus species was assessed in the rhizospheres of four cultivars of sorghum sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg/ha). Two cultivars (IS 5322-C and IS 6320) demanded the higher amount of nitrogen to grow, whereas the other two (FBS 8701-9 and IPA 1011) did not. Using the DNA extracted from the rhizospheres, a Paenibacillus-specific PCR system based on the RNA polymerase gene (rpoB) was chosen for the molecular analyses. The resulting PCR products were separated into community fingerprints by DGGE and the results showed a clear distinction between cultivars. In addition, clone libraries were generated from the rpoB fragments of two cultivars (IPA 1011 and IS 5322-C) using both fertilization conditions, and 318 selected clones were sequenced. Analyzed sequences were grouped into 14 Paenibacillus species. A greater diversity of Paenibacillus species was observed in cultivar IPA 1011 compared with cultivar IS 5322-C. Moreover, statistical analyses of the sequences showed that the bacterial diversity was more influenced by cultivar type than nitrogen fertilization, corroborating the DGGE results. Thus, the sorghum cultivar type was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the habitats investigated.
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PMID:Diversity of Paenibacillus spp. in the rhizosphere of four sorghum (Sorghum bicolor) cultivars sown with two contrasting levels of nitrogen fertilizer assessed by rpoB-based PCR-DGGE and sequencing analysis. 1805 Dec 96

Human type B synoviocytes are involved in joint injury during rheumatic diseases by producing inflammatory mediators such as interleukin-6 (IL-6). The increased level of purine and pirimidine nucleotides in the synovial fluid of rheumatoid arthritis (RA) patients could activate the large family of P2 receptors. Thus, we investigated the presence of P2 receptors in human type B synoviocytes from rheumatoid joints, also evaluating whether the P2X7 receptor is involved in IL-6 release. Reverse transcriptase polymerase chain reaction analysis revealed messenger ribonucleic acid (mRNA) expression for the P2X1, P2X2, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14 but not the P2X3, P2Y2, and P2Y6 receptors. The expression of the P2X7 receptor was confirmed by Western blot analysis. Adenosine triphosphate (ATP) and the P2X7 receptor agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) triggered an increase in intracellular calcium, thereby suggesting the expression of functional P2 receptors, including the P2X7 receptor. Moreover, BzATP treatment upregulated both IL-6 mRNA and protein expression. Synoviocytes spontaneously released low quantities of IL-6; the incubation with BzATP induced the release of larger amounts of the cytokine, and such a release was blunted by the P2X7 antagonist oxidized ATP. The selective P2X1 and P2X3 receptor agonist alpha,beta-methylene ATP did not affect IL-6 release. Finally, BzATP failed to induce a significant uptake of the large-molecule YO-PRO, thus suggesting the lack of pore formation after P2X7 receptor stimulation. In conclusion, among the different P2 receptors expressed on human RA type B synoviocytes, the P2X7 receptor may modulate IL-6 release but not inducing changes in cell membrane permeability.
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PMID:Human rheumatoid synoviocytes express functional P2X7 receptors. 1854 80

Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet the mechanistic features of this evolution remain elusive. Here, we compared the transcriptional landscapes of the distantly related budding and fission yeast. We adapted the Precision Run-On sequencing (PRO-seq) approach to map the positions of RNA polymerase active sites genome-wide in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Additionally, we mapped preferred sites of transcription initiation in each organism using PRO-cap. Unexpectedly, we identify a pause in early elongation, specific to S. pombe, that requires the conserved elongation factor subunit Spt4 and resembles promoter-proximal pausing in metazoans. PRO-seq profiles in strains lacking Spt4 reveal globally elevated levels of transcribing RNA Polymerase II (Pol II) within genes in both species. Messenger RNA abundance, however, does not reflect the increases in Pol II density, indicating a global reduction in elongation rate. Together, our results provide the first base-pair resolution map of transcription elongation in S. pombe and identify divergent roles for Spt4 in controlling elongation in budding and fission yeast.
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PMID:Divergence of a conserved elongation factor and transcription regulation in budding and fission yeast. 2737 Dec 23

We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.
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PMID:Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). 2744 63

The genome-wide identification of microRNA transcription start sites (miRNA TSSs) is essential for understanding how miRNAs are regulated in development and disease. In this study, we developed mirSTP (mirna transcription Start sites Tracking Program), a probabilistic model for identifying active miRNA TSSs from nascent transcriptomes generated by global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq). MirSTP takes advantage of characteristic bidirectional transcription signatures at active TSSs in GRO/PRO-seq data, and provides accurate TSS prediction for human intergenic miRNAs at a high resolution. MirSTP performed better than existing generalized and experiment specific methods, in terms of the enrichment of various promoter-associated marks. MirSTP analysis of 27 human cell lines in 183 GRO-seq and 28 PRO-seq experiments identified TSSs for 480 intergenic miRNAs, indicating a wide usage of alternative TSSs. By integrating predicted miRNA TSSs with matched ENCODE transcription factor (TF) ChIP-seq data, we connected miRNAs into the transcriptional circuitry, which provides a valuable source for understanding the complex interplay between TF and miRNA. With mirSTP, we not only predicted TSSs for 72 miRNAs, but also identified 12 primary miRNAs with significant RNA polymerase pausing alterations after JQ1 treatment; each miRNA was further validated through BRD4 binding to its predicted promoter. MirSTP is available at http://bioinfo.vanderbilt.edu/mirSTP/.
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PMID:Identification of active miRNA promoters from nuclear run-on RNA sequencing. 2846 90

Coupling molecular biology to high-throughput sequencing has revolutionized the study of biology. Molecular genomics techniques are continually refined to provide higher resolution mapping of nucleic acid interactions and structure. Sequence preferences of enzymes can interfere with the accurate interpretation of these data. We developed seqOutBias to characterize enzymatic sequence bias from experimental data and scale individual sequence reads to correct intrinsic enzymatic sequence biases. SeqOutBias efficiently corrects DNase-seq, TACh-seq, ATAC-seq, MNase-seq and PRO-seq data. We show that seqOutBias correction facilitates identification of true molecular signatures resulting from transcription factors and RNA polymerase interacting with DNA.
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PMID:Universal correction of enzymatic sequence bias reveals molecular signatures of protein/DNA interactions. 2912 7

Isopropyl alcohol and ethyl alcohol have been used as low-level disinfectants in healthcare settings for many years. Recent studies have found that ethyl alcohol inhibits protein synthesis in Escherichia coli by direct effects on ribosomes and RNA polymerase and that 60%-70% solutions have in vitro efficacy against murine norovirus, Ebola virus, and several coronaviruses. Alcohol prep pads or towelettes containing isopropyl or ethyl alcohol and water have been used primarily for disinfection of small noncritical items due to a concern regarding their rapid evaporation rates and associated short contact times. Sterile alcohol solutions are used mostly for disinfection of compounding pharmacies and controlled areas. One new Environmental Protection Agency (EPA)-registered cleaner/disinfectant formulation differs from other alcohol-based disinfectants by virtue of having a 30-second contact time for multiple pathogens and a toxicity rating of category IV. Multiple disinfectants containing ethyl alcohol and/or isopropyl alcohol combined with other active agents such as quaternary ammonium or phenolic compounds are widely used for disinfecting environmental surfaces in healthcare facilities. Infect Control Hosp Epidemiol 2018;39:323-328.
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PMID:Alcohols as Surface Disinfectants in Healthcare Settings. 2937 3


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