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)

The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.
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PMID:Purification and molecular structure of RNA polymerase from influenza virus A/PR8. 235 36

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.
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PMID:Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase. 238 51

Myxococcus xanthus, a myxobacterium, contains a peculiar branched RNA-linked DNA called msDNA. Reverse transcriptase has been shown to be required for the production of msDNA. Existence of proteins that bind to one of the two msDNAs in M. xanthus, msDNA.Mx162, was examined by gel retardation assays. Total cell-free extract yielded two distinct retarded bands. Both bands were sensitive to treatment with proteinase K, indicating that there is a protein(s) that is able to bind to msDNA. Further, the formation of the bands was inhibited by the addition of nonradioactive msDNA but not by a large excess of poly(dA) in the presence of a 5000-fold excess of poly(dI.dC).poly(dI.dC). In vivo footprinting using dimethyl sulfate revealed that the deoxynucleotide stretch from 60 to 161 is protected. When a M. xanthus cell lysate was centrifuged in a 16-30% glycerol gradient, msDNA was found to sediment in two peaks: a major peak corresponding in size to 14 S, and a minor one at 5 S. These results indicate that msDNA.Mx162 exists as a complex with specific proteins in the cell.
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PMID:Myxococcus xanthus msDNA.Mx162 exists as a complex with proteins. 250 5

The in vitro transcription reaction of bluetongue virus (BTV) is characterized by a core-mediated, temperature-dependent inhibition at high core concentrations and temperatures (Van Dijk & Huismans, 1980; Huismans, Van Dijk & Els, 1987a). It has been found that this inhibition is reversible and that an inactivated transcriptase reaction mixture can be reactivated by lowering the temperature of the reaction from 37 degrees C to 28 degrees C. In the same way it is possible to inactivate a reaction by increasing the incubation temperature from 28 degrees C to 37 degrees C. It was also found that the inhibition is counteracted by the addition of sucrose or glycerol. At relatively low core concentrations and in the presence of sucrose it is possible to obtain conditions under which transcription at 37 degrees C is more efficient than at 28 degrees C. The latter conditions probably reflect much better the in vivo temperature optimum for the BTV transcriptase than the in vitro conditions at very high core concentrations.
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PMID:The identification of factors capable of reversing the core-mediated inhibition of the bluetongue virus transcriptase. 283 1

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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PMID:Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. 303 83

The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis.
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PMID:L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. 435 10

Reverse transcriptase is detected in the nuclei as well as the cytoplasm of normal uninfected Japanese quail embryonic tissues. At least 60% of the nuclear enzyme occurs in the free-form, whereas 85% of the cytoplasmic enzyme is bound to particles having a buoyant density equivalent to retroviral cores (1.19-1.22 g/ml). The enzyme is purified by ion-exchange chromatography and glycerol sedimentation gradients. It prefers oligo(dG) . poly(C) in Mg2+ to all other synthetic template-primers, and transcribes poly(A) containing RNAs. Information present in the above particles is endogenous indicating that RT is not derived from exogenous retrovirus.
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PMID:Demonstration of free reverse transcriptase in the nuclei of embryonic tissues of the Japanese quail. 619 28

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.
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PMID:Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. 625 35

An RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.
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PMID:RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties. 768 22

Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.
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PMID:Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes. 803 Dec 33


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