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 effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.
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PMID:P2Y(11) receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors. 1152 39

The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Delta phenotypes. In particular, an rtf1Delta paf1Delta double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Delta single mutant. In addition, expression of the G(1) cyclin CLN1, reduced nearly threefold in paf1Delta, is restored to wild-type levels in the rtf1Delta paf1Delta double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.
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PMID:Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex. 1188 86

TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1-7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1-6). Unlike TOT1-3 (ELP1-3) and TOT5-7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.
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PMID:Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action. 1192 32

The effects of parathyroid hormone (PTH) on tension and intracellular Ca level ([Ca ] ) were examined in ring preparations of rat mesenteric artery using isometric tension recording and the fura-2 method, respectively. The PTH (30 n ) elicited relaxation in arterial rings precontracted by phenylephrine regardless of the presence or absence of endothelium. In the endothelium-denuded arterial rings precontracted by 3 micro M of phenylephrine or 60 m of potassium chloride (KCl), PTH-related protein and PTH produced concentration-dependent relaxation to the same extent, but inhibited contraction induced by phenylephrine more effectively than that induced by KCl. Phenylephrine-induced tonic contraction was changed to a phasic one with decreased peak tension in the presence of PTH. Similar changes were observed with extracellular Ca removal or methoxyverapamil plus SK&F96365, respective of voltage-gated and receptor-operated Ca channel inhibitors. Phenylephrine evoked a concentration-dependent contraction concomitant with an increase in [Ca ]. PTH reduced both responses to the same extent. In a Ca -free solution, PTH inhibited a phasic contraction and a transient increase in [Ca ] in response to phenylephrine but not caffeine. Reverse transcriptase-polymerase chain reaction showed that PTH and PTH receptors were expressed in the rat mesenteric artery. In this tissue, PTH increased cyclic adenosine monophosphate (cAMP) levels. These results suggest that the inhibitory effect of PTH on alpha -adrenoceptor-mediated contraction results from the inhibition of Ca influx through receptor-operated and voltage-gated Ca channels, and Ca release from Ca stores, probably via increased cAMP in the rat mesenteric artery.
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PMID:Relaxant mechanisms of parathyroid hormone in rat mesenteric artery. 1235 17

The human elongator complex was found to be very similar to the yeast Elongator both in composition and its interaction with RNA polymerase II. But little is known about its functions in vivo. In this study, we analyzed the functions of the help3, the catalytic subunit of human Elongator, using a yeast complementation system. The results indicated that help3 was able to significantly complement the growth defects of the elp3delta yeast strain under high temperature and caffeine conditions. Gene expression analysis showed that help3 significantly resumed the slow activation of the pho5 gene under the low phosphate condition, and when heat shocked it increased the expression level of ssa3 gene. The yhelp3 containing the HAT domain of human elp3 and the remainder of yeast elp3 had a higher complementation ability than human elp3, while the yhelp3HA T- with the catalytic HAT domain deleted had no complementation ability. These results implicate that human Elp3 subunit had similar functions to those of the yeast, and the HAT activity of human Elp3 was essential for its function.
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PMID:[The elp3 subunit of human elongator complex ignificantly complemented the growth defects of yeast elp3delta strain]. 1636 16

To analyze the local conformational changes of RNA molecules, we developed a site-specific fluorescent labeling method for RNA fragments by T7 transcription, using unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y) and between 2-amino-6-(2-thiazolyl)purine (v) and y. Ribonucleoside 5'-triphosphates of 5-fluorescence-linked y derivatives can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Using this specific transcription, the substrate of a fluorescein-linked y was introduced into a theophylline-binding RNA aptamer. The replacement of U6 by the fluorescein-linked y maintained both the binding ability and selectivity of the aptamer to theophylline. Furthermore, the fluorescence intensity was increased upon theophylline binding, but was not changed by the addition of caffeine.
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PMID:Site-specific incorporation of fluorescent probes into RNA by specific transcription using unnatural base pairs. 1715 Jul 46

Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34Delta weakened the binding of Rpa49 to RNA polymerase. rpa34Delta mutants were caffeine sensitive, and the rpa34Delta mutation was lethal in a top1Delta mutant and in rpa14Delta, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49Delta mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34Delta mutants, but strains carrying rpa49Delta and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30 degrees C, failed to grow at 25 degrees C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49Delta and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.
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PMID:Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription. 1808 78

Cellular stress and DNA damage up-regulate and activate p53, fundamental for cell cycle control, senescence, DNA repair and apoptosis. The specific mechanism(s) that determine whether p53-dependent cell cycle arrest or p53-dependent apoptosis prevails in response to specific DNA damage are poorly understood. In this study, we investigated two types of DNA damage, chromium treatment and gamma irradiation (IR) that induced similar levels of p53, but that mediated two distinct p53-dependent cell fates. Chromium exposure induced a robust DNA-dependent protein kinase (DNA-PK)-mediated apoptotic response that was accompanied by the rapid loss of the cyclin-dependent kinase inhibitor 1A (p21) protein, whereas IR treatment-induced cell cycle arrests that was supported by the rapid induction of p21. Inhibition of DNA-PK effectively blocked chromium-, but not IR-induced p53 stabilization and activation. In contrast, inhibition of ATM and ATR by caffeine had the inverse effect of blocking IR-, but not chromium-induced p53 stabilization and activation. Chromium exposure ablated p21 transcription but PUMA and Bax transcription was significantly enhanced compared to non-damaged cells. In contrast, IR treatment triggered significant p21 mRNA synthesis in addition to PUMA and Bax mRNA production. While chromium treatment enhanced the binding of p53 and RNA polymerase II (RNA Pol II) to both the p21 and PUMA promoters, RNA Pol II elongation was only observed along the PUMA gene and not the p21 gene. In contrast, following IR treatment, RNA Pol II elongation was observed on both p21 and PUMA. Chromium-induced apoptosis therefore involves DNA-PK-mediated p53 activation followed by preferential transcription of pro-apoptotic PUMA over anti-apoptotic p21 genes.
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PMID:Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNA-PK) and differential induction of p53 target genes. 1860 74

Porcine cytochrome P450 (CYP) 1A2 was purified to electrophoretic homogeneity from the hepatic microsomes of beta-naphthoflavone-treated male pigs. In a reconstituted system, this enzyme showed a good catalytic activity towards caffeine, acetanilide, and methoxyresorufin, all known markers of mammalian CYP1A2. Using 3'- and 5'-rapid amplification of coding DNA (cDNA) ends (RACE), we amplified from the liver RNA of control pigs a full-length 1827 bp cDNA containing an open reading frame of 1548 bp which encoded a putative CYP1A2 protein of 516 amino acids and an estimated Mr of 58 380 Da. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that the messenger RNA (mRNA) of CYP1A2 was expressed in liver, heart and nasal mucosa but not in lung, small intestine, kidney and brain. Using the pCW vector containing a N-terminal modified cDNA, pig CYP1A2 was expressed in Escherichia coli. 3-[(3-Chloroamidopropyl)dimethylmmonio]-1-propane-sulfonate (CHAPS)-solubilized E. coli preparations expressing CYP1A2 produced a functionally isoform which, in a reconstituted system, was catalytically active toward ethoxyresorufin and methoxyresorufin showing K(m)'s similar to those obtained with CYP1A2 purified from pig liver or human recombinant CYP1A2. Taken together, these results demonstrate that domestic pigs have a functionally active CYP1A2 gene well expressed in the liver with biochemical properties quite similar to those corresponding to the human enzyme.
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PMID:Purification, molecular cloning, heterologous expression and characterization of pig CYP1A2. 1894 57

Thein vitro DNA- or RNA-directed synthesis of the large subunit (LS) of spinach chloroplast ribulose-1,5-biphosphate carboxylase (RuP2C) has been examined in a highly definedE. coli transcription-translation system. Spinach chloroplast DNA, RNA and recombinant plasmids containing the spinach chloroplast LS gene (rbcL) have been used as templates in thein vitro system and a quantitative assay has been developed to measure LS formation. Thein vitro formed product contains formylmethionine at the N-terminal position and sediments primarily as a monomer. There is no detectable enzymatic activity associated with thein vitro product. To determine where theE. coli RNA polymerase used in these systems initiates, we have examined the transcripts produced by this enzymein vitro. Measurements of run-off transcripts indicate thatE. coli RNA polymerase initiates at the same position on the gene as is seenin vivo. In addition, the complete nucleotide sequence of therbcL gene including previously unsequenced 3' and 5' flanking regions has been determined. The sequence agrees, except at two nucleotide positions, with previously published sequencing data for this gene (Zurawski, G, Perrot, B, Bottomley, W, Whitfeld, PR, 1981. Nucleic Acids Res. 9:3251-3270).
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PMID:Synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in anin vitro partially definedE. coli system. 2431 76


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