Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fragment of the SIVmac251 pol gene was expressed in Escherichia coli as a trpE fusion protein. Analysis of extracts from bacteria containing this expression plasmid revealed the presence of a reverse transcriptase activity dependent on Mg2+ as divalent cation and active on both poly(rA).oligo(dT) and poly(rC.oligo(dG) templates. In comparative studies, the SIV and HIV-1 reverse transcriptases expressed in bacteria displayed very similar high sensitivities to the chain terminator inhibitors AZTTP and ddTTP. The reverse transcriptase of Moloney murine leukemia virus and the DNA polymerase of E. coli were both more resistant to ddTTP, and the E. coli enzyme was significantly more resistant to AZTTP.
 ...
PMID:Expression of enzymatically active reverse transcriptase of simian immunodeficiency virus in bacteria: sensitivity to nucleotide analogue inhibitors. 170 May 44

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.
 ...
PMID:Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. 170 16

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.
 ...
PMID:Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate. 170 9

The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.
 ...
PMID:Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs. 171 78

Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5'-triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson-Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding.
 ...
PMID:Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase. 171 63

Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on the avian myeloblastosis virus reverse transcriptase activity. The drug inhibited the DNA synthesis reaction catalyzed by the viral enzyme in the presence of different template-primers. The inhibitory effect by nalidixic acid was higher with polyriboadenylic acid than with polyribocytidylic acid as a synthetic template. With activated DNA as a template nalidixic acid preferentially inhibited the TMP incorporation when compared with the dAMP incorporation. Both these results showed the importance of the presence of adenine in the templates for a more efficient inhibition by nalidixic acid. The inhibition for this drug was also shown in the presence of Mn2+ instead of Mg2+ as the divalent cation, and with a 2'-fluorinated analogue of polyriboadenylic acid as the template. Kinetic data showed a non-competitive inhibition by nalidixic acid in relation to polyriboadenylic acid and to TTP in the reaction catalyzed by reverse transcriptase.
 ...
PMID:Avian myeloblastosis virus reverse transcriptase inhibition by nalidixic acid. 172 88

Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synthesis of long cDNA required 8 mM Mg2+, 4 mM Mn2+, 2 mM dNTPs, and saturating levels of enzyme. Actinomycin D efficiently limited the enzyme to the first strand synthesis. Additional characteristics of the fusion protein are described.
 ...
PMID:Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. 241 Apr 13

The RNA-dependent DNA polymerase of the AIDS-associated retrovirus (ARV) gives highest activity with the synthetic template, poly(rA)oligo(dT) and prefers Mg2+ over Mn2+ as a divalent cation. It can use other template-primer combinations but with poly(rCm)oligo(dG), it prefers Mn2+ over Mg2+. Detection of ARV reverse transcriptase in culture fluids is substantially increased with an optimal KCl concentration and a special combination of EGTA and reducing agents. Our results indicate some distinguishing characteristics of the ARV reverse transcriptase and optimal conditions for its detection.
 ...
PMID:Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. 241 16

Enzymological properties of the RNA-directed DNA polymerase associated with the Suncus murinus mammary tumor virus (Sm-MTV) was investigated and its antigenic relatedness to other retroviral DNA polymerases was examined. The enzyme exhibited higher activity in the presence of Mg2+ than in the presence of Mn2+ with endogenous RNA as well as with almost all of the synthetic template X primers tested. Mg2+ was also effective with poly(2'-O-methylcytidylate) X oligodeoxyguanylate which was known to be specific for Mn2+. To examine the immunological relatedness of this enzyme with other retroviral DNA polymerases, remaining Sm-MTV DNA polymerase activity was measured after treatment of this enzyme with various antisera prepared against each of the reverse transcriptases of Mason-Pfizer monkey virus (MPMV), murine mammary tumor virus (MuMTV), simian sarcoma virus-simian sarcoma associated virus (SSV/SSAV), and Rauscher murine leukemia virus (RLV). No inhibition of the Sm-MTV enzyme activity was observed when treated with the latter three antisera with which the DNA polymerase activities of the corresponding retroviruses were fully inhibited. Only the antiserum against MPMV-enzyme, however, was found to slightly inhibit the Sm-MTV enzyme activity. These results indicate that Sm-MTV DNA polymerase has similar enzymological properties to those of MPMV and MuMTV and shares some common antigenic determinant group(s) with MPMV DNA polymerase.
 ...
PMID:Biochemical and immunological characterization of Suncus murinus mammary tumor virus DNA polymerase. 241 16

The reverse transcriptase from AIDS virus, HTLV-III, was purified and characterized. The purified enzyme has a very high affinity for template primers (rC)n X (dG)12 and (rCm)n X (dG)12 compared to that for (rA)n X (dT)12. In addition, the HTLV-III reverse transcriptase was able to transcribe (rAm)n X (dT)12 very efficiently. The ionic requirements are unique in the sense that HTLV-III reverse transcriptase prefers Mg2+ as divalent ions to transcribe (rC)n X (dG)12 and (rA)n X (dT)12. The Mr of the enzyme is 95 000-98 000. Unlike the HTLV-I reverse transcriptase, the HTLV-III enzyme is highly stable and has a much higher activity in the presence of (rC)n X (dG)12; the Vmax for HTLV-III reverse transcriptase is several-fold higher than that for HTLV-I enzyme. The enzyme activity of the purified reverse transcriptase from HTLV-III was resolved into two peaks on a preparative isoelectric column, one at pH 5.75 and the other at pH 6.25. This leads us to conclude that the reverse transcriptase of HTLV-III is biochemically heterogeneous.
 ...
PMID:Biochemical heterogeneity of reverse transcriptase purified from the AIDS virus, HTLV-III. 241 71


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>