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 (replicase) of encephalomyocarditis (EMC) virus was found to be closely associated with the smooth membranes of infected BHK-21 cells. An RNA-dependent EMC replicase was extracted from the membranes with 0.15% sodium dodecyl sulfate (SDS) and 1,1,2-trichlorotri-fluoroethane (Genetron 113) and further purified by high-salt dextran-polyethylene glycol phase separation, sievorptive chromatography, and glycerol gradient sedimentation. The enzyme does not manifest strict specificity toward EMC RNA template. It can use also Qbeta RNA, rRNA of BHK cells, or poly(C). SDS-polyacrylamide gel electrophoresis of purified EMC replicase labeled with radioactive methionine revealed that, of all the stable EMC proteins, the enzyme contains predominantly the 56,000-dalton (E) polypeptide.
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PMID:Isolation and properties of the replicase of encephalomyocarditis virus. 0 14

Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through poly- rotein of 200,000 molecular weight. This polyprotein (Pr200(gag-pol)) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to gag proteins recognized Pr200(gag-pol). Pr200(gag-pol) contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Pr145(pol)), 135,000 (Pr135(pol)), and 125,000 (Pr125(pol)) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing tryptic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80(pol)) precipitate by antiserum to RT. Purification of active RT enzyme from virions labeled with [(3)H]methionine showed that p80(pol) was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80(pol)), similar in size to mature viral p80(pol), was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80(pol). Pulse-chase studies showed that Pr80(pol), Pr125(pol), and Pr135(pol) were stable polypeptides, whereas Pr200(gag-pol) and Pr145(pol) were unstable precursors. Pulse-chase studies with the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200(gag-pol) occurred for a short time in the absence of protein synthesis.
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PMID:Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein. 7 22

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.
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PMID:Two methyltransferase activities in the purified virions of vesicular stomatitis virus. 20 77

The free 4S RNA of avian RNA tumor viruses is greatly enriched in one of the four methionine tRNAs of the host cells, tRNA4Met. On the assumption that viral tRNAMet forms are identical to the corresponding tRNAs of mouse or chick cells, the following conclusions were drawn concerning the tRNAMet content of oncornaviruses: (1) tRNAMet species may be compartmentalised within the host cells, and the viral tRNA pool could reflect the cellular compartment in which viral maturation takes place since tRNAMet forms distribute unevenly between different fractions of a cell homogenate. (2) tRNA4Met appears to have no special role in the modulation of protein synthesis in as much as no functional difference between tRNA2Met and tRNA3Met, tRNA4Met could be demonstrated in in vitro protein synthesising systems. (3) tRNA4Met differs in nucleotide sequence from all other host cell tRNAMet forms except possibly tRNA2Met. The nucleotide sequences of two tRNAMet species, tRNA1Met and tRNA4Met, have already been determined and the sequence of another host cell tRNAMet, tRNA3Met, was derived from the analogy of its sequence to that of tRNA4Met since the two molecules differ in only 6 nucleotides out of 76. (4) Avian myeloblastosis virus reverse transcriptase has been shown to bind specifically tRNA4Met and tRNATrp in whole cell tRNA and therefore the free tRNA4Met in the virion particle may exist substantially bound to virion-associated transcriptase.
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PMID:Selection of methionine tRNAs by avian oncornaviruses. 21 69

An RNA-dependent RNA polymerase activity has been demonstrated for spring viremia of carp virus (SVCV). The optimal temperature for in vitro synthesis of RNA was 20 to 25 degrees C. The SVCV enzyme activity was stimulated when the methyl donor S-adenosyl-L-methionine was included in the reaction mixture. S-adenosyl-L-methionine was not particularly effective in stimulating the virion RNA polymerase activity of vesicular stomatitis virus or pike fry rhabdovirus. The 5' nucleotide of the SVCV viral RNA is pppAp.
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PMID:Spring viremia of carp virus RNA and virion-associated transcriptase activity. 56 17

1. Messenger activity of phage f2 RNA modified with methoxyamine under non-denaturing conditions was studied in E. coli-free system. The incorporation of amino acids into phage polypeptides was decreased, and the synthesis of phage-specific proteins was diminished. The RNA replicase synthesis was more affected than synthesis of coat protein. The impaired messenger activity of the methoxyamine-modified f2 RNA was due to the blocking of elongation process by modified cytosines present in RNA chain. 2. Specificity of f2 RNA to stimulate ribosomal binding predominantly at the coat protein initiation site was not affected by methoxyamine-treatment, as demonstrated by unchanged binding of f[3H]Met-tRNA and [14C]alanyl-tRNA to ribosomes. 3. Unfolding of f2 RNA molecule on treatment with methoxyamine in the presence of guanidine-HCl resulted in a significant increase of RNA capacity to direct fMet-tRNA binding to ribosomes. Sucrose-density gradient profiles revealed the formation of polysome-like initiation complexes indicating that ribosomes were able to bind at many hitherto inaccessible initiation codons in RNA molecules. fMet-tRNA bound to ribosomes in the presence of unfolded RNA was found to be fully reactive with puromycin.
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PMID:Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis. 78 15

Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.
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PMID:Nucleotide binding by the poliovirus RNA polymerase. 132 24

A plasmid has been constructed containing the DNA sequences that direct the expression of the aphthovirus RNA-dependent RNA polymerase (virus infection-associated antigen, VIAA) in its native form. The aphthovirus polypeptide was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant protein has been purified and used in enzyme-linked immunoelectrotransfer blots to detect aphthovirus-specific antibodies in the sera of persistently infected animals. Furthermore, studies were carried out to test the hypothesis that antibodies against other nonstructural antigens appear in the sera of these animals. It was established that antibodies against polypeptides 3A and 3B can serve as complementary markers for late aphthovirus-carrier state detection. The considerable potential of this approach to detect aphthovirus-specific antibodies, when the isolation of infectious virus is not possible, was demonstrated. Negative results were obtained in animals from virus-free areas and in vaccinated cattle. This assay has the added advantage that no infectious or noninfectious virus is involved during antigen production.
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PMID:Expression of the aphthovirus RNA polymerase gene in Escherichia coli and its use together with other bioengineered nonstructural antigens in detection of late persistent infections. 165 2

Papaverine, an inhibitor of cAMP phosphodiesterase, reduced yields of infectious vesicular stomatitis virus in HEp-2 cells approximately 100-fold if added to cultures at a concentration of 30 microM before and after virus infection. The extent of papaverine-induced suppression of viral growth was dependent on drug dose and treatment regimen. Cells progressively recovered their viral permissive state after removal of drug. The cyclic nucleotide, cGMP, nullified the inhibitory effect of papaverine if added to cells during drug treatment. Pulse labeling experiments with [35S]methionine showed that papaverine compromises production of all virus-specific proteins in infected cells without adversely affecting host cell protein synthesis. Treatment of cells with papaverine strongly inhibited the production of viral RNA and both cellular RNA and DNA. It was found that VSV causes an immediate but transient stimulation of DNA synthesis in HEp-2 cells which is prevented by papaverine treatment. This drug also selectively blocked primary transcription of VSV in vivo and to a lesser extent in vitro RNA polymerase activity of the virion-bound transcriptase. The finding that papaverine has a strong inhibitory effect on viral biosynthesis including early transcription suggests that VSV replication may depend on host factors that regulate intracellular levels of cyclic nucleotides such as cAMP.
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PMID:Inhibitory effect of papaverine on RNA and protein synthesis of vesicular stomatitis virus. 241 Oct 62

Reverse transcriptase of the avian sarcoma and leukosis retroviruses is a heterodimer composed of a 63-kDa alpha and a 95-kDa beta polypeptide chain, both of which are encoded in the pol gene and are produced by proteolytic processing of a larger precursor. We previously constructed a bacterial expression clone of the entire pol coding region that produces a protein 4 kDa larger than the mature viral beta subunit. By use of this clone and synthetic oligonucleotides to introduce stop codons, two derivatives have been constructed: one that directs synthesis of a protein equivalent to the mature beta subunit and the other that directs synthesis of a protein equivalent to alpha subunit. Predicted amino acid sequences of these proteins differ from their viral counterparts only by an initiator methionine that was added to the N termini for expression in Escherichia coli. Both bacterially expressed proteins exhibit reverse transcriptase activity and appear to function as homodimers. The properties of these proteins resemble those of the viral reverse transcriptase heterodimer; however, the bacterially produced alpha dimer protein could be distinguished from the other proteins by its increased sensitivity to heat inactivation, which also has been reported for the corresponding viral product. These results show that correct folding and expression of enzymatic function does not require formation of a precursor. The alpha and beta clones provide a convenient source of individual pol gene products for further evaluation of their roles in the synthesis and integration of retroviral DNA.
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PMID:The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities. 245 57


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