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)

Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66,000 subunit and its C-terminally truncated form of M(r) 51,000, but, when expressed as a recombinant M(r) 66,000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66,000 and 51,000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66,000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66,000 subunits containing single-point or deletion mutations, with wild-type M(r) 51,000 subunits, and the resulting heterodimers analysed.
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PMID:Formation of heterodimers of human-immunodeficiency-virus-type-1 reverse transcriptase by recombination of separately purified subunits. 137 16

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is comprised of two subunits of approximately 66kD and 51kD. We have defined the carboxyl terminus of the 51kD molecule using the 66kD RT and HIV-1 protease (PR) expressed in yeast. Precise constructs encoding the 66kD and 51kD molecules were expressed individually, in yeast, at high levels. The purified recombinant subunits were shown to associate into heterodimers that retained both RT and RNase H activities. Only the 66kD molecule could associate into homodimers. Such homodimers retained approximately 80% of the RT activity of the heterodimers. Our data demonstrates that the 51/66kD heterodimer, analogous to that found in vivo, can be reconstituted in vitro and is more efficient in both RT and RNase H activity than the homodimer.
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PMID:Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast. 169 61

HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented.
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PMID:HIV reverse transcriptase structure-function relationships. 171 68

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
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PMID:Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. 171 16

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

A recombinant p66 form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69-81] from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV-1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV-1 RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4 degrees C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA-dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV-1 RT upon incubation with the HIV-1 protease. When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4 degrees C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66-containing species were stable and displayed identical RNA-dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole-mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.
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PMID:Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase. Asymmetry in p66 subunits of the p66/p66 homodimer. 751 87

A recombinant homodimer p66/p66 of the HIV-1 reverse transcriptase (RT) was expressed in and purified from a protease-deficient strain of the yeast Saccharomyces cerevisiae. The RNase H activity associated with the homodimer was biochemically characterized. The effect of cations and the hybrid substrate specificity were studied. Some compounds which have been found to inhibit retroviral replication were tested as potential inhibitors of the retroviral DNA polymerase and RNase H activities. Most of these compounds inhibited preferentially the DNA polymerase activity. On the other hand, only suramin was found to inhibit RNase H more efficiently than DNA polymerase. As in the case of the DNA polymerase activity, the thiol-reacting agent N-ethylmaleimide (NEM) did not affect the RNAse H activity of HIV RT. When the effect of NEM was tested against E coli RNase H, a weak inhibitory effect was detected. Surprisingly, NEM strongly inhibits the same bacterial RNase H in the presence of a recombinant form of HIV RT devoid of nuclease activity. These results strongly suggest an interaction between E coli RNase H and HIV-1 RT.
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PMID:The ribonuclease H activity of HIV-1 reverse transcriptase: further biochemical characterization and search of inhibitors. 768 32

We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.
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PMID:Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli. 768 63

The polymerase activity of the p51 homodimeric form of HIV reverse transcriptase was characterized by activity gel analysis, steady-state kinetic measurements, and processivity assays, and the activity was shown to be highly similar to that for the p66/p51 heterodimer. Recombinant 51- and 66-kDa reverse transcriptase proteins were individually expressed from an HIV-1 Pol gene having an accumulation of natural amino acid mutations compared to the BH10 clone (Ratner et al., 1985). The preparation of an active p51 homodimer critically depended on low temperature during its expression in bacterial cultures. Activity gel analysis demonstrates that refolded p51 protein derived from denatured p66/p51 heterodimer yields an active polymerase. The p51 homodimer has approximately one-half the activity and processivity of the heterodimer, while both enzymes have similar thermostability. Steady-state measurements reveal no significant differences in apparent affinities for substrate or homopolymeric template-primer, suggesting that the subunits in both enzyme forms have similar conformations. Template challenge experiments show that the off-rates for template-primer are lower, but as indicated by primer extension analyses, processivity is less for p51 homodimer. These results show that the RNase H domain is not essential for the assembly of the functional polymerase, but suggest that it enhances processivity.
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PMID:HIV-1 reverse transcriptase: polymerization properties of the p51 homodimer compared to the p66/p51 heterodimer. 769 Nov 76

Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2.
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PMID:Characterization of the p68/p58 heterodimer of human immunodeficiency virus type 2 reverse transcriptase. 863 74


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