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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cell-free extract containing TMV-RNA replicase was prepared from TMV-infected tobacco leaves. It could synthesize double-stranded RNAs in the presence of four nucleoside triphosphates (among them, UTP was tritium-labelled), magnesium ion and actinomycin D. It was confirmed by polyacrylamide-agarose gel electrophoresis, RNase treatment, thermal denaturation and self annealing that 3H-ds RNAs, obtained from phenol-SDS extraction and Serva cellulose column chromatography, consisted of replicative form (RF) and replicative intermediate (RI) of TMV-RNA, with molecular weights of 40 X 10(6) and 5.0 X 10(6), respectively. Molecular hybridization competition experiment showed that 60-70% of the nascent RNAs in the 3H-ds RNA were plus strand of tMV-RNA.
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PMID:The nature of the RNA products synthesized in vitro with a cell-free extract from TMV-infected tobacco leaves. 723 51

A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA.
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PMID:Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol. 805 62

Fungal vesicles isolated from a hypovirulent strain (EP113) of the chestnut blight fungus, Cryphonectria parasitica, contained double-stranded RNA and possessed an RNA-dependent RNA polymerase activity which was absent in comparable preparations from dsRNA-free vesicles of a virulent strain (EP 155). RNA polymerase activity remained associated with hypovirulent vesicles when these were sedimented through a 10 to 40% sucrose gradient and the polymerase activity coincided with the peak of dsRNA content. Incorporation of [32P]-UTP into RNA was proportional to the amount of vesicles present in the reaction mixture. Enzyme activity was dependent upon the presence of dsRNA-containing vesicles, Mg2+ and the four ribonucleotide triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The optimum temperature for polymerase activity was 30 degrees, and temperatures higher than 35 degrees inactivated the enzyme. Treatment of vesicles with low concentrations of detergent led to a two- to threefold increase in the rate of RNA synthesis. The RNA polymerase products, synthesized in vitro, hybridized specifically with C. parasitica genomic dsRNAs. Hybridization to single-stranded cDNA clones containing inserts of the terminal domains of the homopolymer and heteropolymer ends of the dsRNA showed that the reaction products were full-length copies of both strands of the dsRNA. Single-stranded RNA synthesis was asymmetrical, with greater than 80% of the polymerase products being of positive polarity. It can be estimated that in the fungal vesicles isolated from hypovirulent C. parasitica, transcription of the dsRNA into mRNA for translation is in the order of two- to eightfold more active than replication. On the basis of our results and of the evidence accumulated so far, we suggest that the replication strategy employed by the hypovirulence-associated dsRNA is following that of positive-strand RNA viruses.
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PMID:Membrane-associated replication of an unencapsidated double-strand RNA of the fungus, Cryphonectria parasitica. 831 9

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
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PMID:Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression. 895 11

Cucumber mosaic virus (CMV) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic RNAs. The very 3' ends of each of the genomic RNAs can fold into a tRNA-like structure. Based on the structural analysis of the 3' tRNA-like structure of the brome mosaic virus (BMV), we superimposed and redrew the 3' tRNA-like structure of CMV. We homogenized virus infected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra-centrifugation three times to get detergent solubilized membrane bound fractions. We accidentally found that these fractions were enriched with a host-encoded RNA-dependent RNA polymerase (RdRp) activity. Similar activity could also be found in other plants tested. Alternately, the membrane bound fraction could be simply precipitated by low speed centrifugation (3,000 g) and high speed ultra-centrifugation (40,000 g). The pellet was then suspended in a detergent-containing buffer, after which 25%-55% glycerol gradient fractionation was performed. Activity was tested through the incorporation of [alpha-32P]UTP using endogenous CMV RNAs as templates on each fraction collected. It was found that most of the fractions contained the viral-encoded RNA-dependent RNA polymerase. The products of RdRp reaction were found to have a double-stranded from through further analysis of the RNase protection assay.
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PMID:The preparation of RNA-dependent RNA polymerase complex from virus infected plants. 961 71

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.
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PMID:Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase. 963 Feb 51

We have previously investigated, in studies of rat distal colonic mucosa, the effect of ATP added to the basolateral side on ion transport and [Ca2+]i. It was demonstrated that ATP acts via a P2Y1 receptor to increase [Ca2+]i and NaCl secretion. In the present study we investigated the effect of luminally added nucleotides (ATP, UTP) on transepithelial voltage (Vte) and resistance (Rte) in Ussing chamber experiments on rat distal colonic mucosa. Both nucleotides induced a rapid and transient (within 30 s) change of Vte to lumen-positive values (resting Vte: -2+/-1 mV; peak Vte after 100 micromol/l ATP: +2.4+/-1.1 mV) and a decrease of Rte from 89. 9+/-10.3 to 83.8+/-9.1 Omegacm2 (n=10). Similar values were obtained with luminal UTP (n=15). The estimated EC50 values for both nucleotides were approximately 6 micromol/l. The ATP-induced Vte effect was nearly completely sensitive to Ba2+. Addition of the K+ channel blocker Ba2+ (1 mmol/l) to the luminal solution reversibly inhibited 77+/-4% (n=5) of the ATP-induced Vte effect. Experiments to identify the respective P2 receptor subtype revealed the following rank order of potency at 500 micromol/l agonist: UTP>/=ATP>>2-methylthio-ATP=ADP>>adenosine> AMP>beta, gamma-methylene-ATP (n=5). This closely resembles the published rank order for the P2Y2 receptor. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique P2Y2 receptor-specific mRNA was detected in total RNA extracted from isolated crypts. In summary these data indicate that luminal ATP and UTP act via a P2Y2 receptor in the luminal membrane of colonic mucosa to elicit a transient K+ secretion.
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PMID:Luminal ATP induces K+ secretion via a P2Y2 receptor in rat distal colonic mucosa. 971 4

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.
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PMID:Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus. 974 Jul 82

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

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.
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PMID:Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP. 1019 56


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