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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

At concentrations of 7 times 10(-6) to 7 times 10(-5) M, derivatives consisting of the polycylic ring structures fluoranthene, fluorenone, fluorene, anthraquinone, xanthenone, and dibenzofuran with appropriate amine side chains inhibited by over 90% the purified RNA-directed DNA polymerase of avian myeloblastosis virus acting on poly(deoxyadenylate-deoxythymidylate) [poly(dA-dT)]. Of these, only the fluoranthene derivatives were strong inhibitors of the viral DNA polymerase directed by polyadenylate-oligodeoxythymidylate [poly(A)-(dT)12-18]. Low levels of fluoranthene derivatives (1 times 10(-5) M) also strongly inhibited polymerase with polyinosinate-oligodeoxycytidylate [poly(I)-(dC)12-18], activated calf thymus DNA, and viral 70S RNA as templates, but not with polycytidylate-oligodeoxyguanylate as template. A comparison of the activity of 11 fluoranthene derivatives with different side chains showed that the structure of the amine side chain influenced both the extent of antipolymerase activity with a given template and the relative inhibition with different synthetic DNA and RNA templates. The naturally occurring polyamines, spermine, spermidine, and putrescine, did not inhibit the activity of the viral DNA polymerase. Studies on the mechanism of action indicated that the synthetic derivatives inhibited polymerase activity by binding to the template and not to the enzyme: 1) inhibition by fluoranthene derivatives was overcome by the addition of excess template including poly(dA-dT), poly(A)-(dT)12-18, poly(I)-(dC)12-18, viral 70S RNA, and activated calf thymus DNA; 2) the degree of inhibition by fluoranthene derivatives was unaffected by the addition of the creased viral DNA polymerase; 3) with the same template, Escherichia coli DNA-directed RNA polymerase and the viral RNA-directed DNA polymerase were inhibited to about the same extent; and 4) the derivatives formed a complex with DNA, poly(I), and poly(A) that was stable to exclusion chromatography on Sephadex G-100. Several derivatives also had biologic activity, since they blocked the ability of the murine sarcoma virus to transform cells.
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PMID:Inhibition of purified DNA polymerase of RNA tumor viruses by fluoranthene derivatives and analogues of tilorone hydrochloride. 5 Oct 87

The size of the DNA product synthesized by RNA-directed DNA polymerase (isolated from avian myeloblastosis virus) was found to be important for complementary DNA (cDNA)-mRNA hybridization reactions. Incomplete cDNA to rabbit reticulocyte globin mRNA formed poor hybrids and presumably lacked sequences needed for hybridization. The size of the cDNA synthesized was influenced by the reaction conditions used. The complementary DNA product contained 10 S material when synthesis was done at high deoxynucleoside triphosphate concentrations (greater than 50 muM) while the product was smaller than the template when synthesis was at lower concentrations. The concentration and size (oligo(dT)6 to (dT)10) of primer had little or no effect on the product size. Increasing the concentration of 10 S globin mRNA caused the cDNA product to contain more small material. The cDNA synthesized at high deoxynucleoside triphosphate concentrations was fractionated into heavy, medium, and light fractions by alkaline sucrose density centrifugation. All hybridized to globin mRNA. The larger cDNAs had a higher TM when hybridized to globin mRNA, a lower dTMP/dCMP ratio (indicating that the poly(dT) region constituted a smaller fraction of the molecule), and gave increased protection of 125I-labeled mRNA from nuclease digestion. The full size cDNA was especially useful for studying the RNA transcribed from chromatin by RNA polymerase. The complement of the 5' end of the mRNA is contained only in full size cDNA; the 5' end is the part of the mRNA first transcribed by the RNA polymerase assuming correct transcription. Thus, full size cDNA can hybridize more effectively to the short RNA transcripts that are obtained than partial cDNA. RNA transcribed from rabbit bone marrow chromatin by Escherichia coli RNA polymerase hybridized twice as efficiently to complete cDNA as it did to partial cDNA demonstrating the usefulness of full size cDNA.
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PMID:Importance of full size complementary DNA in nucleic acid hybridization. 5 64

Reverse transcriptase from foamy virus, strain H4188 was estimated and purified. The enzyme has the following characteristics: 1. The reaction utilized preferentially oligo (dT) poly (rA) as a primer-template; however, the synthetic primer-template oligo (dT) poly (dA) could also be used to some extent. 2. The reaction utilized oligo (dG) poly (rC) as a primer-template with very low efficiency. 3. The crude virus preparation had a detectable endogenous reaction using the four deoxyribonucleotides for DNA polymerization. 4. The cation requirement for the enzyme reaction was much more biased for Mn++ than for Mg++ ions. 5. The molecular weight of the partially-purified enzyme was estimated to be about 80,000. Aggregates of 240,000 daltons were also seen. The activity of this enzyme was not inhibited by antisera against the reverse transcriptases of various type C RNA viruses, namely, feline endogenous leukemia virus, RD 114, Woolly simian sarcoma virus (SSV-1) and avian myeloblastosis virus (AMV). Antiserum against Rauscher leukemia virus (RLV) enzyme was marginally active against foamy virus enzyme, perhaps indicating a slight cross-reaction. The biochemical characteristics of foamy virus reverse transcriptase seemed to be very close to those of the type C RNA viruses, but the immunological reaction proved that the foamy virus reverse transcriptase was distinct from the others.
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PMID:Reverse transcriptase of foamy virus. Purification of the enzymes and immunological identification. 7 44

4'-(9-Acridinylamino)methanesulphon-m-anisidide (AMSA) (NSC 141549), an acridine derivative with activity against a variety of laboratory tumors in vivo, is presently undergoing Phase 1 clinical evaluation. The interaction of AMSA with DNA and its effects on nucleic acid-polymerizing enzymes were examined in an attempt to define the site of cytotoxicity of AMSA. Binding of AMSA to DNA, as demonstrated by equilibrium dialysis and spectrophotometric methods, appears to be similar to other aminoacridines, in that two types of binding sites (type 1 and type 2) were observed. Fluorescence studies and thermal denaturation studies gave strong evidence that AMSA type 1 binding was by intercalation into DNA. The binding of AMSA to DNA was without marked base-pair specificity. Furthermore, the effect of AMSA on nucleic acid-polymerizing enzyme activities (mouse embryo DNA polymerase alpha, avian myeloblastosis virus reverse transcriptase, and Escherichia coli RNA polymerase) was studied. Inhibition of enzyme activity by AMSA appeared to be independent of DNA base sequence. The relatively high concentrations of AMSA required for inhibition of these enzymes as compared to the concentrations of AMSA necessary for cytotoxicity in vitro suggest that the interaction with DNA alone might not fully explain its antitumor activity.
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PMID:Interaction of 4'-(9-acridinylamino)methanesulfon-m-anisidide with DNA and inhibition of oncornavirus reverse transcriptase and cellular nucleic acid polymerases. 7 12

beta-Lapachone is a naturally occuring compound that can be isolated from a number of tropical trees. It is shown to be a potent inhibitor of reverse transcriptase activity from both avian myeloblastosis virus and Rauscher murine leukaemia virus. In addition, it affects eukaryotic DNA-dependent DNA polymerase-alpha activity: 50% inhibition is reached in 60-min incubation time by about 8 micron beta-lapachone. Enzyme activity is inhibited irrespective of the purity of the enzyme used or of the amount or type of template/primer or substrate present. The inhibitory effect of the drug is only observed in the presence of dithiothreitol. The primary site of action of beta-lapachone appears to be the enzyme protein, as is also borne out by the specificity of its action. Eukaryotic DNA-dependent DNA polymerase-beta, prokaryotic DNA-dependent DNA polymerase I, several other nucleic acid polymerases and some completely unrelated enzymes are not affected. Reverse transcriptase and DNA-dependent DNA polymerase-alpha may be in someway related in possessing similarly exposed '--SH structures' in their active sites. beta-lapachone thus affords a novel means of studying such interrelationships and of further characterizing enzymes.
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PMID:beta-Lapachone, an inhibitor of oncornavirus reverse transcriptase and eukaryotic DNA polymerase-alpha. Inhibitory effect, thiol dependence and specificity. 7 23

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

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.
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PMID:Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin. 43 65

The mechanism of the effect of an RNA polymerase II (RNA nucleotidyltransferase II) stimulation factor isolated from the nuclei of chicken myeloblastosis cells was studied. The stimulation requires the presence of all four nucleoside triphosphates and depends upon an exogenous DNA template. In the absence of the factor, RNA synthesis ceases after 20-30 min, but in the presence of the factor, synthesis continues up to 60-80 min. Addition of the factor at 35 min after incubation causes resumption of RNA synthesis. The factor greatly stimulates the activity of RNA polymerase II at low enzyme concentrations. The RNA polymerase activity is more sensitive to alpha-amanitin inhibition when the factor is present. Experiments of [gamma-32P]ATP incorporation reveal that the factor provides for an increased frequency of initiation of RNA chains, both of the primary initiation events and re-initiation after previous ones were completed. A slightly higher rate of RNA chain growth was also observed with this factor but the ultimate size of RNA synthesized was not affected, as determined by formaldehyde/sucrose gradient centrifugation. These data suggest that the factor functions at the initiation stages of the RNA polymerase reaction.
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PMID:Increased frequency of initiation of RNA synthesis due to a protein factor from chicken myeloblastosis nuclei. 105 84

Reverse transcriptase template switching has been invoked to explain several aspects of retroviral replication and recombination, and has been reported in vitro for the Moloney murine leukemia virus (M-MuLV) reverse transcriptase. During in vitro cDNA synthesis, the avian myeloblastosis virus (AMV) reverse transcriptase can switch from one template to another in a homology-dependent and temperature-dependent manner. Chimeric cDNA molecules are generated within 30 min at high incubation temperatures, with an increasing efficiency from 42 degrees C to 50 degrees C. Such products are detectable only after much longer incubation times when primer extension reactions are carried out at lower temperatures (90 min at 37 degrees C).
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PMID:Temperature-dependent template switching during in vitro cDNA synthesis by the AMV-reverse transcriptase. 127 21

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
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PMID:Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. 168 52


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