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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two DNA strand transfer reactions occur during retroviral reverse transcription. The mechanism of the first, minus strand strong-stop DNA, transfer has been studied in vitro with human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) and a model template-primer system derived from the HIV-1 genome. The results reveal that HIV-1 RT alone can catalyze DNA strand transfer reactions. Two kinetically distinct ribonuclease (RNase) H activities associated with HIV-1 RT are required for removal of RNA fragments annealed to the nascent DNA strand. Examination of the binding of DNA.RNA duplex and single-stranded RNA to HIV-1 RT during strand transfer supports a model where the enzyme accommodates both the acceptor RNA template and the nascent DNA strand before the transfer event is completed. The polymerase activity incorporated additional bases beyond the 5' end of the RNA template, resulting in a base misincorporation upon DNA strand transfer. Such a process occurring in vivo during retroviral homologous recombination could contribute to the hypermutability of the HIV-1 genome.
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PMID:Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. 127 6

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
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PMID:Mechanism and fidelity of HIV reverse transcriptase. 128 79

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.
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PMID:Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease. 156 48

The handle region (residues 84-99) in ribonuclease HI (RNase HI) from Escherichia coli, which is rich in basic amino acid residues, was altered by alanine-scanning mutagenesis. Fifteen mutant proteins were purified to homogeneity and analyzed for the enzymatic activity. A mutation of either of 2 tryptophan residues at 85 or 90 resulted in a large increase in the Km value along with a large decrease in the Vmax value. These values probably resulted from conformational changes introduced by the mutations as indicated by the CD spectra of these mutant proteins. All other mutant enzymes had Vmax values similar to that of the wild-type enzyme. In contrast, replacement of any basic amino acid residue in the handle region, except for lysine 86, yielded proteins whose Km values were 3-5-fold higher than the wild-type enzyme. Such effects were shown to be cumulative, suggesting strongly that the cluster of positive charges in the handle region is important for the effective binding of the substrate. Interestingly, the region of human immunodeficiency virus reverse transcriptase with homology to E. coli RNase HI lacks the handle region which may account for the poor RNase H activity of the domain when separated from the polymerase domain.
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PMID:Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate. 164 12

The role of direct virus infection as a determining factor in acquired immunodeficiency syndrome (AIDS) dementia was investigated using in situ hybridisation for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV). Four of the five AIDS dementia patients in this series demonstrated HIV infected cells distributed in widely different parts of the brain, but only one case showed HCMV infected cells. The greater abundance of HIV was in subcortical white matter in nodular areas consisting of monocyte/macrophage infiltrates. The cells were occasionally arranged as a multinucleated syncitium. In two cases, a few large cells with the appearance of neurons were positive for HIV hybridisation. By appropriate treatment with ribonuclease, it was shown that hybridisation was primarily to HIV RNA. HCMV infected cells were observed in small numbers in only one of the positive cases, suggesting that HCMV is not a determining factor in AIDS dementia. HCMV positive cells were located in the grey matter, with an appearance suggestive of neurons. Cells expressing the MHC-class II antigen HLA-DR, a marker of reactive microglia and macrophages, were observed to be extensive in affected brain sections in the one case examined. These cells were present in greater number than HIV infected cells. In this case, extensive numbers of HIV infected cells were noticed along the peripheral margin of the substantia innominata. This could indicate infection in this case of a critical brain region from the cerebrospinal fluid.
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PMID:Examination of brains of AIDS cases for human immunodeficiency virus and human cytomegalovirus nucleic acids. 254 95

The genome of human immunodeficiency virus encodes a protein that dramatically elevates amounts of viral proteins. The precise mechanism of this trans-activation remains to be established. It has been reported that trans-activation can occur without major changes in the levels of mRNA. We constructed recombinant plasmids containing those viral sequences required in cis for trans-activation linked to the chloramphenicol acetyltransferase gene. These plasmids were introduced into cultured cells in either the presence or absence of a second plasmid that directed expression of the viral trans-activator protein. Expression of the chloramphenicol acetyltransferase gene was measured at the level of protein (by enzymatic assay) and RNA (by ribonuclease protection and primer extension). Our results demonstrate that trans-activation is accompanied by large increases in mRNA levels; these increases may be sufficient to explain the elevated levels of trans-activated protein.
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PMID:Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. 302 48

A homology has been found between an octapeptide involved in attachment of the human immunodeficiency virus to helper/inducer T cells and an octapeptide segment of bovine pancreatic ribonuclease A. This segment (residues 19-26) contains the sites for subtilisin cleavage of this enzyme into the S-peptide and S-protein. From the X-ray crystal structure of ribonuclease, this sequence is known to be exposed to solvent and interacts little with the rest of the protein. A structure for the human immunodeficiency virus attachment peptide can be deduced from this homology, as a well-defined structure has been determined for this sequence in ribonuclease. This can be readily accomplished using previously developed computer methods based upon conformational energy calculations. The calculated structure for human immunodeficiency virus peptide is identical to the ribonuclease segment (19-26) in backbone conformation. It is stabilized by internal interactions of nonpolar residues, and by exposure of polar hydroxyl groups. The results suggest that the T-cell human immunodeficiency virus receptor may be hydrophilic in nature and that conservation of the sequence in two presumably functionally unrelated proteins is related to the need for conservation of exposed structure.
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PMID:A strong homology exists between the active T-cell binding gp120 octapeptide of human immunodeficiency virus and the subtilisin cleavage peptide of bovine ribonuclease A. 303 Mar 17

After infection of the respective target cells with the human immunodeficiency virus (HIV-1) viral progeny is produced only after a short temporary delay of some days, depending on cell type. After this period of time a sudden onset of HIV-1 protein synthesis with a dramatic increase in virus release occurs. (2'-5')Oligoriboadenylates [(2'-5')A], capable to activate a latent ribonuclease (RNase L) degrading both mRNA and rRNA, are known mediators involved in the early response of cells to virus infection. Here we show that the (2'-5')A-synthesizing (2'-5')A synthetase, which is inducible by interferon and activated by double-stranded RNA, as well as a (2'-5')A nuclease (2',3'-exoribonuclease) are associated with the nuclear matrix of uninfected and infected H9 cells, also in the absence of interferon. Infection of H9 cells with HIV-1 was found to cause a strong (7.7-fold) enhancement of (2'-5')A synthetase activity and a smaller (2-fold) increase of 2',3'-exoribonuclease activity. Simultaneously the concentration of synthesized (2'-5')A increased 5 to 10 times in isolated nuclei. After incubation for 2 to 3 days both enzyme activities reached a maximum and then dropped below their initial values. Concomitantly a drastic increase in virus production occurred, as judged by reverse transcriptase activity in the culture fluid. These results suggest that the (nuclear matrix-associated) (2'-5')A system might be important during the initial stage of HIV infection, also by destructing matrix-bound viral messengers.
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PMID:Alteration of nuclear (2'-5')oligoriboadenylate synthetase and nuclease activities preceding replication of human immunodeficiency virus in H9 cells. 322 94

Three human cell lines of astrocytic origin were evaluated for expression of a human T-lymphocyte surface glycoprotein, T4, which also serves as a cellular receptor for the human immunodeficiency virus (AIDS virus, HIV). T4 antigen was detected on the cell surface of 2 of these cell lines using monoclonal OKT-4 antibody and flow cytometry. Gene transcripts encoding the T4 molecule were detected by a ribonuclease protection assay in surface T4-positive and -negative cells. Our results suggest that astrocytes may serve as targets for HIV infection in the brain.
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PMID:Expression of the T4 molecule (AIDS virus receptor) by human brain-derived cells. 349 19

Capillary electrophoresis with laser-induced fluorescent detection, a one-dimensional version of the well-established planar analytical method of polyacrylamide gel electrophoresis, has been proven to be a powerful new microanalytical method for profiling complex carbohydrates. In this paper a comparison is presented between the planar high concentration polyacrylamide gel electrophoresis method and capillary electrophoresis of different carbohydrates with respect to performance and efficiency. N-Linked oligosaccharides were released from several glycoproteins, including fetuin, human immunodeficiency virus (HIV) envelope recombinant glycoprotein (GP-120), alpha 1-acid glycoprotein and ribonuclease B, using recombinant peptide-N-glycosidase F (PNGase F). Both separation methods involve labeling of the released carbohydrates at the reducing end with the fluorescent dye, disodium 8-amino-1,3,6-naphthalene trisulfonate (ANTS). Fluorophore labeling was followed by separation of the labeled oligosaccharides either by high concentration polyacrylamide gel electrophoresis or capillary electrophoresis.
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PMID:Capillary and slab gel electrophoresis profiling of oligosaccharides. 749 47


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