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)

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.
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PMID:Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties. 383 93

Reverse transcriptase isolated from avian myeloblastosis virus (AMV) and Rauscher murine leukemia virus (RLV) were examined for their ability to catalyze polymerization, ribonuclease H, pyrophosphate exchange, and pyrophosphorolysis reactions. A detailed characterization and a study of requirements for the expression of pyrophosphate exchange and pyrophosphorolysis reactions indicated that a variety of RNA and DNA template-primers supported these catalytic reactions. Furthermore, hydrogen bonding of template to primer was essential, although RNA:RNA template-primers, e.g. poly(rA) . (rU)9 or 70 S RNA . tRNA complex, were not utilized for these reactions. AMV enzyme required Mg2+, and RLV enzyme Mn2+, as the preferred divalent metal ion for the expression of these activities. Response of various catalytic reactions to site-specific inhibitors revealed that polymerization and pyrophosphate exchange reactions were susceptible to reagents that affected either the substrate or the template binding site, intrinsic zinc, or sulfhydryl groups. RNase H and pyrophosphorolysis activities, on the other hand, exhibited susceptibility only to the template site-specific reagent. We, therefore, conclude that RNase H and pyrophosphorolysis reactions are catalyzed through the template binding site while polymerization and pyrophosphate exchange reactions require additional participation of the substrate binding site, as well as that of intrinsic zinc and the presence of reactive sulfhydryl groups.
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PMID:Enzymatic activities associated with avian and murine retroviral DNA polymerases. Catalysis of and active site involvement in pyrophosphate exchange and pyrophosphorolysis reactions. 615 89

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 template-dependent polyuridylic acid [poly(U)] polymerase has been isolated from BHK cells infected with foot-and-mouth disease virus (FMDV). Enzyme activity in a 20,000 x g supernatant of a cytoplasmic extract was concentrated by precipitation with 30 to 50% saturated ammonium sulfate. The poly(U) polymerase was freed of membranes by sodium dodecyl sulfate and 1,1,2-trichlorotrifluoroethane extraction, and RNA was removed by precipitation with 2 M LiCl. The solubilized poly(U) polymerase required polyadenylic acid as template complexed to an oligouridylic acid primer and Mg2+ for activity, but was inhibited by Mn2+. Antisera from animals infected with FMDV had previously been shown to inhibit the activity of FMDV RNA replicase complexed to the endogenous RNA template. The same antisera also inhibited the activity of poly(U) polymerase. Antisera depleted of antibody by absorption with the virus infection-associated antigen of FMDV no longer inhibited replicase and polymerase activities. The evidence suggests that FMDV RNA replicase, poly(U) polymerase, and the virus infection-associated antigen share a common protein.
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PMID:Isolation of a foot-and-mouth disease polyuridylic acid polymerase and its inhibition by antibody. 625 Dec 48

A soluble RNA-dependent RNA polymerase was purified from the cytoplasm of poliovirus-infected HeLa cells. A single virus-specific protein designated as p63 (or NCVP4) copurified with this activity. The purified polymerase was free of ribonuclease activity and was shown to copy poliovirion RNA when oligo(U) was added to the in vitro reaction mixture. Characterization of the product RNA by electrophoresis in methylmercury (II) hydroxide-agarose gels showed that genome-sized copies of poliovirion RNA were synthesized in vitro by the purified polymerase. The product RNA was shown to be heteropolymeric, complementary to virion RNA, and covalently linked to oligo(U). The product RNA contained the expected distribution of UMP and GMP containing dinucleotide pairs which included a very low frequency of CpG pairs. The amount, size distribution, and rate of synthesis of product RNA was very dependent on the in vitro reaction conditions. Full sized product RNA was synthesized in about 6 min when reaction conditions were used that yielded maximum elongation rates (pH 8.0, 7 mM Mg2+, 37 degrees C). Under these conditions, most of the product RNA recovered from a 1-h reaction was full sized. Thus, the polymerase was found to specifically initiate synthesis at the 3'-end of the template using an oligo(U) primer and to carry out an elongation reaction at about 1250 nucleotides/min that resulted in the synthesis of full sized product RNA.
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PMID:Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase. 627 40

Effects of Ca2+ and Mg2+ on the "switch-on" of transcriptase function in reovirus intermediate subviral particles were examined. Switch-on in this system is triggered by K+ ions, whereas Na+ ions antagonize the effects of K+ ions. Once effected, switch-on is irreversible. In the absence of Ca2+ and Mg2+ ions, switch-on of transcriptase is effected by incubation at 37 degrees C in the presence of a particular ratio of K+/Na+ (total K+ plus Na+ being kept constant). Inclusion of Ca2+ in the activation step lowers the ratio that triggers the switch-on event. This indicates that Ca2+ destabilizes the switched-off state of the transcriptase. Inclusions of Mg2+ ions in the activation step raises the K+/Na+ ratio necessary to trigger switch-on. Thus Mg2+ tends to stabilize the switched-off state of the transcriptase to the triggering action of K+ ions. Inclusion of both Ca2+ and Mg2+ together during the activation step results in mutual inhibition of the effects expected from each divalent cation alone. These results indicate that switch-on of transcriptase function, which is triggered by K+ ions, can be modulated by interactions involving the major physiological cations (Na+, K+, Mg2+, Ca2+).
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PMID:Effects of Ca2+ and Mg2+ on the switch-on of transcriptase function in reovirus in vitro. 642 61

RNA-dependent RNA polymerase from healthy tomato plant tissue accepts potato spindle tuber viroid (PSTV) RNA as a template for the in vitro synthesis of full-length RNA copies of the PSTV genome. Viroid transcription requires the presence of Mn2+ and /or Mg2+ ions and is not inhibited by concentrations of 10(-5) M alpha-amanitin. This is the first report of a well-defined product synthesized in vitro by an RNA-dependent RNA polymerase from healthy plants.
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PMID:In vitro transcription of viroid RNA into full-length copies by RNA-dependent RNA polymerase from healthy tomato leaf tissue. 689 6

The basophilic leucaemia cell line RBL-2H3 exhibits a robust inwardly rectifying potassium current, IKIR, which is likely to be modulated by G proteins. We examined the physiological and molecular properties of this KIR conductance to define the nature of the underlying channel species. The macroscopic conductance revealed characteristics typical of classical K+ inward rectifiers of the IRK type. Channel gating was rapid, first order (tau approximately 1 ms at -100 mV) and steeply voltage dependent. Both activation potential and slope conductance were dependent on extracellular K+ concentration ([K+]o) and inward rectification persisted in the absence of internal Mg2+. The current was susceptible to a concentration- and voltage-dependent block by extracellular Na+, Cs+ and Ba2+. Initial IKIR whole-cell amplitudes as well as current rundown were dependent on the presence of 1 mM internal ATP. Perfusion of intracellular guanosine 5'-Q-(3-thiotriphosphate) (GTP[gamma S]) suppressed IKIR with an average half-time of decline of approximately 400 s. It was demonstrated that the dominant IRK-type 25 pS conductance channel was indeed suppressed by 100 microM preloaded GTP[gamma S]. Reverse transcriptase-polymerase chain reactions (RT-PCR) with RBL cell poly(A)+ RNA identified a full length K+ inward rectifier with 94% base pair homology to the recently cloned mouse IRK1 channel. It is concluded that RBL cells express a classical voltage-dependent IRK-type K+ inward rectifier RBL-IRK1 which is negatively controlled by G proteins.
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PMID:Physiological and molecular characterization of an IRK-type inward rectifier K+ channel in a tumour mast cell line. 760 35

To estimate the polyamine distribution in Escherichia coli, the binding constants (K) for DNA, RNA, phospholipids, and ATP were calculated under the condition of 10 mM Tris-HCl, pH 7.5, 150 mM K+, and 10 mM Mg2+. The binding constants of spermidine for E. coli DNA, E. coli 16S rRNA, phospholipids in E. coli membrane, and ATP were 0.015, 0.066, 0.028, and 0.081 mM-1, respectively. Similarly, those of putrescine were 0.010, 0.010, 0.007, and 0.037 mM-1, respectively. The concentrations of putrescine, spermidine, and ATP and phosphates in DNA, RNA, and phospholipids in E. coli harvested at A600 = 0.3 were 32.2, 6.88, and 2.66 and 96.4, 436, and 57.2 mM, respectively. Accordingly, the percentage of spermidine bound to DNA, RNA, phospholipids, and ATP and that of free spermidine were 5.1, 90, 0.7, 0.8, and 3.8%, respectively. The percentage of putrescine bound to DNA, RNA, phospholipids, and ATP and that of free putrescine were 9.3, 48, 1.4, 2.6, and 39%, respectively. The results indicate that most spermidine exists as a spermidine--RNA complex, and about 40% and 50% of putrescine exists as a free form and a putrescine--RNA complex in cells, respectively. Under the conditions that the synthesis of specific proteins such as RNA replicase is stimulated by polyamines in a cell-free system, the amount of spermidine and putrescine bound to RNA was close to the value estimated in cells. Experiments to demonstrate the polyamine stimulation of MS2 RNA-directed RNA replicase synthesis in vivo were thus performed, and the results were confirmed.
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PMID:Estimation of polyamine distribution and polyamine stimulation of protein synthesis in Escherichia coli. 767 29

The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity. 785 86


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