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 effect of proteins soluble in acidic chloroform-methanol (ACMS proteins) on the transcriptase activity of virus ribonucleoproteins (RNPs) in vitro has been studied. Experiments with ACMS membrane (M) proteins from type A and B orthomyxoviruses, as well as from vesicular stomatitis virus, showed that inhibition of the viral RNP transcriptase activity occurred when they interacted with M proteins isolated from viruses of a different serotype, or even of a different family. The presence of ACMS proteins capable of inhibiting the transcriptase activity of orthomyxovirus RNP in vitro was also detected in human blood plasma and among proteins produced by human leukocytes. Determination of the minimum concentration of M protein inhibiting the RNP transcriptase activity, and analysis of the fowl plague virus M protein-RNP complex formed in the in vitro system, showed that the M protein was capable of inhibiting RNP transcriptase activity at a M:RNP ratio of 0.1 to 0.2:1.
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PMID:In vitro inhibition of negative strand virus transcriptase activity by proteins soluble in acidic chloroform-methanol. 630 Feb 85

The transcriptase activity of influenza A virus ribonucleoproteins was inhibited by 42 to 49% in vitro in the presence of membrane (M) protein. The addition of M protein to the system of ribonucleoprotein preparations isolated from rimantadine-sensitive or rimantadine-resistant influenza virus strains, as well as the addition of M protein isolated from a sensitive strain, in the presence of rimantadine further inhibited the transcriptase activity of such complexes by approximately 40%. In the system containing the same ribonucleoprotein preparations, but with M protein isolated from a rimantadine-resistant influenza virus strain, the transcriptase activity was not sensitive to rimantadine. The data show that M protein can influence the activity of influenza A virus virion transcriptase and that the susceptibility of influenza virus to rimantadine may be due to the peculiarities of M protein.
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PMID:Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine. 689 43

The ribonucleoprotein core of reovirus is a multienzyme complex that transcribes messenger ribonucleic acid (mRNA) from double-stranded RNA templates. So far, the core has resisted attempts to disassemble it and identify the polypeptide species responsible for RNA polymerase activity. As an alternative approach, we tested pyridoxal 5-phosphate (PLP) as a potential affinity labeling reagent for reovirus transcriptase in vitro; PLP has been used as an affinity reagent for cellular and viral nucleic acid polymerases. We found that PLP inhibited reovirus transcriptase reversibly (apparent Ki = 0.2 mM), but the inhibition was noncompetitive with respect to each of the four ribonucleoside triphosphates. This interaction required both the aldehyde and phosphate moieties in PLP, since pyridoxamine and pyridoxal were relatively inactive. To identify the polypeptides involved, we labeled the PLP--core complex by reductive alkylation with [3H]borohydride. At PLP concentrations close to the apparent Ki, labeling was selective for the two largest virion polypeptides, lambda 1 and lambda 2. At saturation, there were only 10 high-affinity PLP binding sites per core in each of the lambda polypeptide species. These findings implicate either or both lambda polypeptide species in viral transcription and they indicate that a special population, representing no more than 10% of the total lambda molecules in each core, participates in RNA synthesis.
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PMID:Pyridoxal phosphate as a probe of reovirus transcriptase. 735 41

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.
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PMID:Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase. 748 31

To elucidate the mechanism of transcription and replication of Sendai virus, we developed an efficient and faithful in vitro transcription system using purified virus particles. The in vitro RNA synthesis was almost entirely dependent on the addition of eukaryotic cell extracts, including those from various cultured mammalian cells, mammalian tissues, and even from plant cells. The RNA products were almost identical to authentic mRNA species synthesized in the infected cells, in their size distribution, the presence of 3'-poly(A) tail and the presence of methylated 5'-cap structure (m7GpppAm). Ribonuclease protection experiments after annealing the in vitro RNA with viral genomic RNA (vRNA) indicated that the virion-associated RNA-dependent RNA polymerase transcribes correct regions of the RNA genome in vitro. The active component(s) that is required for Sendai virus mRNA synthesis was partially purified from bovine brain and was separated into at least two complementary fractions, one of which could be replaced by highly purified cellular tubulin. When viral ribonucleoprotein complexes were used instead of virus particles in the in vitro transcription, only Sendai virus-infected cell extracts supported mRNA synthesis, and extracts from uninfected cells or cells infected with other viruses were found to be inert. These results suggest that, in addition to the general factors which are present ubiquitously in eukaryotic cells, a factor(s) specific to Sendai virus-infection is required for Sendai virus transcription.
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PMID:Protein factors required for in vitro transcription of Sendai virus genome. 762 18

Lyssaviruses are considerably adapted to neural tissue, although they can also be replicated in muscle and glandular cells. In neural tissue their reproduction takes place almost exclusively in neurons, and in the course of their dissemination they make use of the structural peculiarities of this highly differentiated cell type. The replication takes place completely in the cytoplasm, although rhabdovirus leader RNA enters the nucleus and by blocking host DNA and RNA synthesis promotes viral synthetic processes. In the cytoplasm the two phases of viral reproduction, the synthesis of nucleocapsids and the formation of the envelope together with the assembly of the virion, are separate in time and space. By this separation the transmission of infection by the incomplete form of the virus, i.e., by the synaptic transfer of ribonucleoprotein-transcriptase complexes is also possible. The formation of viral envelope and assembly of full viruses on the cisternal system of the host neurons is a highly complex process, as presented here in a three-dimensional analysis. Due to the high complexity of virus assembly, defects in construction are frequent, accounting for the high yield of defective interfering particles in the course of the reproduction of lyssaviruses.
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PMID:Reproduction of lyssaviruses: ultrastructural composition of lyssavirus and functional aspects of pathogenesis. 785 98

Influenza virus polymerase complexes that were expressed in the absence of genomic viral RNA and nucleoprotein were examined for endonuclease activity and transcriptase ability in vitro. Nuclear extracts of cells that express influenza virus polymerase through recombinant vaccinia virus infection did not display specific endonuclease activity in vitro. This polymerase presumably represents an early form of enzyme present in infected cells prior to ribonucleoprotein assembly. Upon addition of a virus-like model RNA template, containing the partially complementary sequence found at the ends of viral RNA, endonuclease activity is stimulated in a concentration-dependent and sequence-specific manner. Once stimulated, the polymerase is able to elongate from the added viral template. Thus, addition of viral template is required for polymerase activity, while the presence of nucleoprotein is not required for limited transcription. Also, full activation of this recombinant viral polymerase is dependent on the presence of both the 3' and 5' ends of the viral genome, as model RNA containing either end alone could not effectively trigger the endonuclease.
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PMID:Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease activity. 810 13

The nucleoside analog 2'-deoxy-2'-fluoroguanosine (2'-fluorodGuo) is phosphorylated by cellular enzymes and reversibly inhibits influenza virus replication in chick embryo cells within the first 4 h of infection. RNA hybridization studies revealed that primary and secondary transcription of influenza virus RNA were blocked at a compound concentration of 10 microM, but no inhibition of cell protein synthesis was seen even at high compound concentrations (200 microM). In vitro, the triphosphate of 2'-fluorodGuo is a competitive inhibitor of influenza virus transcriptase activity from disrupted virus, with a Ki of 1.0 microM. The cellular polymerases DNA polymerase alpha and RNA polymerase II were only weakly inhibited or were insusceptible to 2'-fluorodGTP. In kinetic studies with the influenza virus transcriptase, 2'-fluorodGTP, in the absence of GTP, blocked elongation of the virus RNA chain. Similarly, by using purified ribonucleoprotein complexes it was found that the addition of a single nucleotide of 2'-fluorodGTP to the virus RNA caused chain termination, which resulted in the blockage of further virus transcription. Furthermore, the specificity for influenza virus transcriptase was confirmed when the transcriptase from partially resistant virus was found to be 10-fold less susceptible to 2'-fluorodGTP (Ki = 13.1 microM).
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PMID:Inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine. 858 25

Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3'-hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex.
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PMID:Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs. 863 70

Influenza A virus RNA-dependent RNA polymerase, purified from virion ribonucleoprotein particles and from which endogenous genomic RNA (vRNA) has been depleted by treatment with micrococcal nuclease, was used to study transcription initiation, elongation, and termination in vitro. Templates that contained either minus- or plus-sense influenza virus nucleoprotein minigenes with conserved 5' and 3' termini and the uridylate tract were constructed. The dinucleotide ApG and alfalfa mosaic virus RNA4 (AlMV4) were used as primers. ApG primed the synthesis of full-length positive-strand or cRNA products and shorter transcripts, depending upon the molar ratio between the nucleoprotein and the vRNA template. Sequence analysis of the ends of these transcripts demonstrated that the 5' termini of both transcripts and the 3' terminus of the full-length product were complementary to the 3' and 5' termini of the vRNA template, respectively, whereas the 3' terminus of the incomplete product corresponded to a sequence located 40 bases downstream from the 5' terminus of the template and was about 20 nucleotides downstream from the uridylate tract, which is the putative signal for polyadenylation. Binding of the cap structure of AlMV4 by the polymerase activated RNA synthesis by ligation-elongation of small genomic RNA fragments which were likely derived from a genome segment protected by the polymerase from micrococcal nuclease digestion. The sequence of these fragments mapped to a region 14 to 28 nucleotides upstream of the 3' terminus of the viral genome. Polymerase subunit involvement in transcription initiation with ApG or AlMV4 was characterized by studying the effect of purified polyclonal antisubunit immunoglobulins of the G class (IgGs) in transcription assays. These results showed that anti-PB2 IgG inhibited transcription initiation in both ApG- and AlMV4-primed reactions, whereas anti-PB1 antibodies also blocked transcription initiated with AlMV4. The differences observed in product size, product sequence, and differential inhibition by antisubunit IgGs are discussed. These observations would support the notion that the influenza virus RNA-dependent RNA polymerase undergoes a conformational change after the binding of the cap structure of host cell heterogeneous nuclear RNA by PB2, which then usually leads to endonucleolytic cleavage of the capped primer 13 nucleotides downstream from the cap.
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PMID:Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro. 864 63


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