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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biochemical characteristics of the RNase H activity associated with immunoaffinity purified human immunodeficiency virus (HIV) reverse transcriptase (RT) were examined. Glycerol gradient centrifugation of HIV RT resulted in a single peak of RNase H, associated with RT activity, with an apparent molecular weight of 110,000. HIV RNase H exhibited a marked substrate preference for poly(dC).[3H]poly(rG) compared to poly(dT).[3H]poly(rA). It did not hydrolyze single-stranded RNA or the DNA component of DNA.RNA hybrids. Products of the HIV RT-associated RNase H reaction consisted primarily of monomers, dimers, and trimers with 3' OH groups. This reaction was Mg2+ dependent, with greater than 90% of maximum activity at MgCl2 concentrations between 4 and 12 mM. The optimum KCl concentration for HIV RT catalyzed polymerization with a poly(rA).(dT)10 template. The optimum pH for HIV RNase H activity was between 8.0 and 8.5, in contrast to an optimum pH of 7.5 to 8.0 for HIV RT activity. The association of RNase H activity with the p66 component of HIV RT was demonstrated by activity gel analysis. These results indicate that HIV RT has an integral RNase H activity; however, some of its properties are different from those of RNase H associated with other retroviral RT's, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization.
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PMID:Human immunodeficiency virus reverse transcriptase-associated RNase H activity. 246 65

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.
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PMID:Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. 247 90

Human immunodeficiency virus reverse transcriptase (HIV-RT) exhibits a strong sensitivity to pyridoxal 5'-phosphate (PLP), a substrate-binding site directed reagent for DNA polymerases (Modak, M. J. (1976) Biochemistry 15, 3620-3626). Treatment of HIV-RT with PLP followed by sodium borohydride reduction of the enzyme-PLP adduct results in irreversible inactivation of polymerase activity while RNase H activity associated with HIV-RT is minimally affected. Kinetic studies indicate that the PLP inhibition is complex. Yet one of the sites of PLP action appears to be involved in the process of dNTP binding as judged by (a) competitive mode of inhibition and (b) blockage of PLP into enzyme protein by the addition of substrate dNTP. Furthermore, this site is the only PLP reactive site which is accessible to borohydride reduction. Comparative tryptic peptide mapping of enzyme treated with PLP under a variety of conditions permitted the identification of a PLP reactive site containing peptide. Furthermore, reactivity of this site was also blocked by inclusion of substrate dNTP and appropriate template-primer. The amino acid composition and sequence analysis of this peptide showed that a lysine residue present at position 263 in the primary amino acid sequence of HIV-RT is the site of PLP reactivity. We therefore conclude that lysine 263 serves as an important part of the dNTP-binding domain in HIV-RT.
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PMID:Substrate binding in human immunodeficiency virus reverse transcriptase. An analysis of pyridoxal 5'-phosphate sensitivity and identification of lysine 263 in the substrate-binding domain. 247 Jul 47

A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy--that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H, including circular poly(A).linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNA.RNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes.
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PMID:Ribonuclease H activities associated with viral reverse transcriptases are endonucleases. 247 Nov 88

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)/ribonuclease H has been expressed to high levels in Escherichia coli from a recombinant plasmid constructed using the polymerase chain reaction (PCR) for in vitro mutagenesis. Translational initiation and termination codons were introduced by the PCR at points corresponding to sites of cleavage of the RT from the gag-pol precursor polyprotein by the HIV-1 protease; the HIV-1 protease is not expressed from this construct. Most of the RT coding sequences derived from PCR were exchanged for a DNA fragment cloned by standard methods to minimize the possibility that an unwanted mutation was introduced during the in vitro amplification. The RT is expressed in bacteria from this plasmid as 66 and 51 kDa proteins, has both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities, and is indistinguishable from native HIV-1 RT in electrophoretic mobility and immunoreactivity. Peptide sequencing of the amino terminus of the HIV-1 RT purified from bacterial lysates is also presented. A novel activity gel assay was used to confirm that only the 66 kd protein catalyzes the RNase H reaction; this assay will simplify analysis of this catalytic activity. This HIV-1 RT expression plasmid is of interest because of the high level of expression in bacteria and the demonstrated RNase H activity of the enzyme. This plasmid will be distributed for research purposes through the NIH AIDS Repository and will facilitate enzymologic, structural, and immunologic evaluation of reverse transcription and its chemotherapeutic inhibition.
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PMID:HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. 247 33

The reverse transcriptase (RT) activity of human immunodeficiency virus type 1 and other retroviruses is closely associated with a hybrid-degrading RNase H activity which is essential for retroviral replication. We have analyzed the effect of sulfated polysaccharides on human immunodeficiency virus type 1 recombinant RT and RNase H activities in vitro. Heparin, dextran sulfates, and xylan polysulfate were found to be much more potent inhibitors of RNase H than of RT and exhibit 50% infective doses of 0.04 to 0.1 micrograms/ml (corresponding to 0.1 to 25 nM) which is up to 5,000-fold more efficient than that for RT. Inhibitors of RNase H activity are attractive as antiviral drugs.
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PMID:Inhibition of human immunodeficiency virus type 1 RNase H by sulfated polyanions. 247 74

A primary site for initiation of plus strand DNA synthesis in human immunodeficiency virus (HIV) corresponds to a 19-nucleotide-long purine rich sequence located just upstream of the U3 region, designated the polypurine tract (PPT). The HIV reverse transcriptase (RT) uses its RNase H activity to cut the genomic RNA after minus strand DNA synthesis. A plus strand PPT primer is formed, extended, and then removed. In vitro, the HIV-RT recognizes this primer specifically, using it much more efficiently than other RNA primers. However, the PPT still primes significantly less efficiently than DNA primers. The 19-nucleotide PPT primer is partially resistant to degradation when compared with other oligoribonucleotides. Prior to initiation of DNA synthesis, several nucleotides are removed by the RT from the 3' ends of some of the PPT primers. Cleavage is enhanced in the absence of dNTPs. We suggest that DNA synthesis suppresses primer degradation, so that primer extension and cleavage occur in proper sequence. As a result of 3' end degradation, PPT elongation products contain 5'-RNA segments from 16 to 19 nucleotides in length. These shorter segments are also generated from a longer transcript containing the PPT sequence, indicating that they are not created as a result of binding of the RT to the 5' end of the PPT oligoribonucleotide. Full-length and shorter versions of the PPT primers are cleaved from the extended DNA by RT. These experiments show that HIV-RT has a specificity to generate a primer in the region of the PPT but that the ends of the primer are not well defined.
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PMID:Use of an oligoribonucleotide containing the polypurine tract sequence as a primer by HIV reverse transcriptase. 749 8

The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptor templates. In reactions performed in the absence of acceptor template, a prominent DNA synthesis product 75 nucleotides in length resulting from pausing DNA synthesis within the homology zone was observed. Prominent donor RNA degradation products of 47 or 54 nucleotides were also observed, in reactions with 80 or 150 mM KCl, respectively. The lengths indicated a potential 13- or 20-nucleotide long, respectively, complementary region between the DNA and RNAs. The 54-, but not the 47-, nucleotide RNA was susceptible to Escherichia coli RNase H, indicating that the DNA was annealed only to the 54-mer. When acceptor was added, a portion of the 75-nucleotide DNA was chased into transfer product at both salt concentrations, and a portion of the 54-mer RNA became resistant to E. coli RNase H. Evidently, this donor RNA was annealed to the 75-nucleotide long DNA but could be actively displaced by the acceptor. Overall, these observations support two mechanisms for transfer. In one, the pause site-specific DNA dissociates from the donor template before transferring. In the other, the acceptor actively displaces the DNA from the donor.
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PMID:The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer from internal regions of heteropolymeric RNA templates. 750 52

A contribution of the 51-kDa subunit of human immunodeficiency virus type-1 reverse transcriptase to activities of the parental heterodimer (p66/p51) was assessed in "selectively deleted" heterodimers whose p51 component contained C-terminal truncations of 13, 19, or 25 residues. Analyses included (i) efficiency of reconstitution into heterodimer, (ii) retention of polymerase and ribonuclease H (RNase H) function, and (iii) interaction with the HIV replication primer, tRNA(Lys,3). Our data suggest that these features of heterodimer reverse transcriptase can be modulated by the extent of the C-terminal p51 deletion. Severely impaired tRNA binding in a selectively deleted heterodimer whose 51-kDa subunit lacks 13 residues, despite retention of enzymatic functions, strengthens arguments for p51 involvement in tRNA binding.
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PMID:Modulation of HIV-1 reverse transcriptase function in "selectively deleted" p66/p51 heterodimers. 750 7

A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV-1) to digestion by the HIV-1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440-Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227-14232). The COOH-terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483-Leu484 and Tyr532-Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436-Ala437 and at Phe440-Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.
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PMID:Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease H as substrates of the viral protease. 750 54


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