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)

Two types of particles were isolated during purification of rotavirus. Dense (D) particles have a density of 1.38 in CsCl and exhibit spontaneously a fully active endogenous transcriptase. Light (L) particles (density of 1.36 in CsCl) need to be treated with chelating agents to show a polymerase activity. The activation process of L particles was studied under strictly controlled monovalent, divalent, and hydrogen ion concentrations. These experiments demonstrate that i) activation is not affected by the ionic strength ii) activation occurs only at a pH higher than 7.1 iii) a low concentration of chelating agent (40 muM EDTA) is sufficient to activate the enzyme. Treatment of particles with EGTA, which chelates selectively Ca2+, leads to unmasking even in the presence of magnesium, indicating that the concentration of free calcium ions plays a major role in the activation process. Various glycosidases, detergents, and chelating agents were tested in respect to unmasking properties. Of these compound only chelating agents turned out to be efficient. Following activation, two glycopeptides were solubilized. These glycopeptides have an apparent molecular weight of 34,000 and 31,000 daltons and react with concanavalin A. The role of Ca2+ upon the stability of virus particles, and the activation of the endogenous transcriptase in vitro and in the infected cells is discussed.
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PMID:Activation of rotavirus RNA polymerase by calcium chelation. 4 4

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.
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PMID:Segments of influenza virus complementary RNA synthesized in vitro. 62 84

The structure of a very common RNA hairpin, 5'GGAC(UUCG)GUCC, has been determined in solution by NMR spectroscopy. The loop sequence, UUCG, occurs exceptionally often in ribosomal and other RNAs, and may serve as a nucleation site for RNA folding and as a protein recognition site. Reverse transcriptase cannot read through this loop, although it normally transcribes RNA secondary structure motifs. A hairpin with that loop displays unusually high thermodynamic stability; its stability decreases when conserved nucleotides are mutated. The three-dimensional structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR using distance geometry, followed by restrained energy minimization. The structure was well-defined despite the conservative use of interproton distances, by constraining the backbone conformation by means of scalar coupling measurements. A mismatch G.U base pair, with syn-guanosine, closes the stem. This hairpin has a loop of only two nucleotides; both adopt C2'-endo sugar pucker. A sharp turn in the phosphodiester backbone is stabilized by a specific cytosine-phosphate contact, probably a hydrogen bond, and by stacking of the cytosine nucleotide on the G.U base pair. The structural features of the loop can explain the unusual thermodynamic stability of this hairpin and its sensitivity to mutations of loop nucleotides.
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PMID:Structure of an unusually stable RNA hairpin. 170 37

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

Base selection by the Qbeta-replicase and E. coli transcriptase has been studied using substrate analogs. Six analogs with normal hydrogen-bonding sites were polymerized by both enzymes. Three analogs containing ring substitutions which affect the conformation at the glycosyl bond would not substitute for their normal congeners. The remaining analog was an excellent substrate for the transcriptase but not the replicase.The results imply that the base selection procedure has stringent requirements for substrate conformation. Part of the restriction may derive from the requirement that template and substrates conform to the stereochemical constraints of a double helix. In the Qbeta-replicase reaction, synthesis in the presence of substrate analogs displayed abortive kinetics, implying that the replicase reaction can be uniquely curtailed by substrate analogs.
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PMID:Q-beta-replicase and E. coli transcriptase: requirements for substrate selection as revealed by a study of base analogs. 491 89

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

The family Bunyaviridae comprises over 200 viruses (serotypes, subtypes, and varieties) that infect vertebrates and/or invertebrates. Four genera of viruses have been defined (Bunyavirus, Nairovirus, Phlebovirus, and Uukuvirus). The main characteristics of the member viruses are: (i) the virus particles are for the most part uniformly spherical, 80-110 nm in diameter, and possess a unit membrane envelope from which protrude polypeptide spikes 5-10nm long; (ii) the viruses have three helical nucleocapsids, often in the form of supercoiled circles, each consisting of a single species of single-stranded RNA, major nucleocapsid polypeptide, N, and at least in some cases minor amounts of a large polypeptide which may be a transcriptase component; (iii) the genome is composed of three species of RNA (L, large; M, medium; and S, small), organized in end-hydrogen bonded circular structures; (iv) most viruses have three major virion polypeptides (N, and two surface polypeptides, designated G1 and G2); (v) for at least some member viruses, the virions have been shown to contain an RNA-directed RNA polymerase, believed to be responsible for the synthesis of viral complementary mRNA, so that bunyaviruses are considered to be negative-stranded viruses; (vi) at least some bunyaviruses are capable of heterologous virus genome segment reassortment and can form recombinant viruses at high or low frequency; (vii) viruses appear to mature primarily at smooth membrane surfaces and accumulate in Golgi vesicles and saccules, or nearby; (viii) transovarial, venereal and/or transstadial transmission in arthropods has been shown to occur for some members of the family.
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PMID:Bunyaviridae. 616 2

Capped ribopolymers lacking a sequence complementary to the common 3' end of the influenza virion RNA segments effectively stimulated transcription of these RNAs by the virion-associated transcriptase. Thus, stimulation of transcription results not from hydrogen bonding between the capped RNA and the 3' end of the virion RNA but presumably from a specific interaction of the capped RNA with protein(s) in the transcriptase complex. Although no specific nucleotide sequence was required for priming activity, capped mRNAs with diminished secondary structure were preferred as primers. Inosine-substituted or bisulfite-modified capped reovirus mRNAs were at least 3- to 5-fold more effective as primers than were the native capped mRNAs. On the other hand, inosine substitution or bisulfite treatment of the uncapped form of reovirus mRNAs converted them from essentially inactive species to potent inhibitors of the transcriptase reaction primed by either ApG or globin mRNA. These effects of reduced secondary structure also most probably reflect an interaction of the exogenous RNAs with transcriptase protein(s). The results obtained from screening a series of native uncapped ribopolymers were consistent with inhibitory activity requiring the absence of most hydrogen bonding in the ribopolymer and also suggested that specific structural feature(s) of the nucleotides in the chain were important.
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PMID:Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA. 693 19

The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.
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PMID:Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns. 754 38

Stimulated B-lymphocytes, isolated from patients with chronic lymphocytic leukemia of B-cell type (B-CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B-lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5-lipoxygenase products. Several thiol-reactive compounds such as N-ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bis[dimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)-PCR analysis demonstrated the expression of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase mRNA in B-CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B-CLL cell membrane. Furthermore, MK886, the FLAP-binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5-HETE formation. Immunocytochemistry showed that 5-lipoxygenase was mainly localized in the nuclei of non-activated B-CLL cells, tonsillar B-lymphocytes and monoclonal B-cells. In contrast, neither human peripheral T-lymphocytes nor Jurkat cells were stained. These results suggest that 5-lipoxygenase and its products function in the nucleus of B-lymphocytes.
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PMID:Studies on the regulation and localization of 5-lipoxygenase in human B-lymphocytes. 755 68


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