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 double-stranded RNA bacteriophage phi 6 contains a virion-associated RNA-dependent RNA polymerase complex. Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase complex, capable of synthesizing RNA strands of positive polarity. The in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation were investigated. Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, activated plus strand synthesis. GTP was the preferred NTP, but dGTP, ddGTP, and the noncleavable analog GMP-PCP could also switch on transcription. This NTP-binding site is probably different from that of the unspecific viral NTPase found in protein P4 and also from that of the rNTP-specific RNA polymerase active site. Binding of purine NTPs was sufficient for the switch-on; hydrolysis of the NTP was not required. Besides nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis. Magnesium ions are required for the activity but calcium ions inhibit the reaction. Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exceptionally high level plus strand synthesis.
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PMID:In vitro transcription of the double-stranded RNA bacteriophage phi 6 is influenced by purine NTPs and calcium. 788 44

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

Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, alpha-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single-stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs.
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PMID:Characterization of an RNA-dependent RNA polymerase activity associated with La France isometric virus. 903 61

Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.
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PMID:Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals. 955 61

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

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site. This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
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PMID:Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension. 974 51

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.
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PMID:Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. 988 74

The recently cloned rabbit kidney Ca2+-sensing receptor (RabCaR) was functionally characterized in microperfused rabbit cortical thick ascending limb (CTAL) segments. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed that this nephron segment contains mRNAs coding for the RabCaR. Elevation of the extracellular Ca2+ concentration ([Ca2+]e) from 1 to 5 mmol l-1 induced an increase in the fluorescence emission ratio (R), thus reflecting an increase in intracellular Ca2+ activity ([Ca2+]i). This increase was inhibited by verapamil, nifedipine and SKF 96365, and potentiated by a previous application of Bay K 8644. Neither verapamil nor Bay K 8644 modified the resting [Ca2+]i. This suggests that the basolateral Ca2+ influx induced by a high [Ca2+]e occurs via verapamil- and dihydropyridine-sensitive Ca2+ channels, which are not open under resting conditions. In contrast to that evoked by antidiuretic hormone (ADH), the [Ca2+]i increase induced by a high [Ca2+]e did not result from an accumulation of inositol phosphates. Neomycin, Gd3+, Mg2+, commonly used agonists of the Ca2+-sensing receptor, did not increase the [Ca2+]i. In the presence of verapamil, ADH still produced a transient [Ca2+]i increase that was not observed in the presence of an increased [Ca2+]e. These results suggest that the RabCaR in rabbit CTAL cells is not functionally coupled to phospholipase C. In conclusion, the high [Ca2+]e-induced [Ca2+]i increase involves verapamil- and dihydropyridine-sensitive Ca2+ channels and is independent of phosphoinositide metabolism. Whether these channels are activated by the RabCaR remains to be elucidated.
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PMID:The Ca2+-sensing receptor in the rabbit cortical thick ascending limb (CTAL) is functionally not coupled to phospholipase C. 1008 49

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U)15) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32 degrees C and pH 8.0. The addition of 10 mmol of Mg2+ increased the activity. The importance of three motifs conserved in RdRp among other positive-strand RNA viruses was confirmed by introduction of an Ala residue to every amino acid of the motifs by site-directed mutagenesis. All mutants lost RdRp activity, but retained the RNA binding activity, except one mutant at Thr287/Asn291. Deletion mutant analysis indicated that the N-terminal region of NS5B protein was critical for the RNA binding. Inhibition of RdRp activity by (-)beta-L-2', 3'-dideoxy-3'-thiacytidine 5'-triphosphate (3TC; lamivudine triphosphate) and phosphonoacetic acid (PAA) was observed after screening of nucleoside analogs and known polymerase inhibitors. These data provide us not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy.
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PMID:Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding. 1009 69

The influenza virus RNA-dependent RNA polymerase protein complex contains an associated RNA endonuclease activity, which cleaves host mRNA precursors in the cell nucleus at defined positions 9-15 nucleotides downstream of the cap structure. This reaction provides capped oligoribonucleotides, which function as primers for the initiation of viral mRNA synthesis. The endonuclease reaction is dependent on the presence of divalent metal ions. We have used a number of divalent and trivalent metal ions alone and in combination to probe the mechanism of RNA cleavage by the influenza virus endonuclease. Virus-specific cleavage was observed with various metal ions, and maximum cleavage activity was obtained with 100 microM Mn2+ or 100 microM Co2+. This activity was about 2-fold higher than that observed with Mg2+ at the optimal concentration of 1 mM. Activity dependence on metal ion concentration was cooperative with Hill coefficients close to or larger than 2. Synergistic activation of cleavage activity was observed with combinations of different metal ions at varying concentrations. These results support a two-metal ion mechanism of RNA cleavage for the influenza virus cap-dependent endonuclease. The findings are also consistent with a structural model of the polymerase, in which the specific endonuclease active site is spatially separated from the nucleotidyl transferase active site of the polymerase module.
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PMID:Metal ion catalysis of RNA cleavage by the influenza virus endonuclease. 1022 Mar 50


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