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

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

Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.
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PMID:Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site. 169 72

The polymerase (P) gene of hepadnaviruses encodes a large polypeptide that appears to participate in several steps in the viral life cycle: packaging of viral RNA, providing the primer for synthesis of minus-strand DNA, synthesizing minus-strand DNA from an RNA template and plus-strand DNA from a DNA template, and degrading viral RNA in RNA-DNA hybrids. To assist in the assignment of these functions to domains of the duck hepatitis B virus polymerase protein, we have constructed a series of substitution mutations and a large insertion mutation, based in part on amino acid sequence comparisons with other proteins known to exhibit reverse transcriptase (RT) and RNase H activities. We found that changes in highly conserved sequences in putative RT and RNase H domains in the carboxy-terminal half of the protein dramatically reduced synthesis of both strands of viral DNA without major effects on RNA packaging into subviral cores. Thus we can uncouple RNA packaging and DNA synthesis but cannot separate RT and RNase H activities as has been done with human hepatitis B virus. The viability of a mutant with a large insertion (123 amino acids) upstream of the RT and RNase H domain indicates that a hinge region may separate parts of the polymerase protein implicated in priming and polymerization.
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PMID:Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase. 169 97

The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the lambda PL promoter. The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion-exchange and size exclusion chromatography. The purified protein appears to be in a native-like homogeneous conformational state as determined by 1H-NMR spectroscopy and circular dichroism measurements. HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441.
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PMID:Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli. 169 94

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.
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PMID:Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli. 170 48

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.
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PMID:HIV-1 reverse transcriptase: structure predictions for the polymerase domain. 170 98

Human immunodeficiency virus reverse transcriptase.RNase H (RT.RNase H) has an RNA hydrolysis specificity which was influenced both by the sequence of the DNA primer-RNA template and by binding of the polymerase active site to the primer 3' terminus. RT.RNase H selectively hydrolyzed the phosphodiester bond between the 15th and 16th ribonucleotide back from the ribonucleotide that is complementary to the primer 3'-terminal deoxynucleotide. The cleavage site for RT.RNase H remained a fixed distance behind the 3'-primer terminus as the polymerase extended the primer. This cleavage was not strongly affected by the position of the DNA primer on the template nor was it affected by reducing the primer length from 40 to 25 nucleotides. These results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 15-16-nucleotide DNA-RNA heteroduplex. Since one helical turn is approximately 10 nucleotides, the distance between the active sites is 1.5 helical turns of heteroduplex. Therefore, the two active sites (catalyzing reactions on opposite strands) bind the same side of the RNA-DNA double helix. RT.RNase H also showed some sequence dependence for the site of hydrolysis. This sequence dependence has not been fully characterized. The rate of RT.RNase H cleavage was weakly inhibited by the next coded deoxynucleoside triphosphate following the incorporation of a dideoxynucleotide.
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PMID:Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and RNA hydrolysis activities. 170 25

The mode of action of the RNase H activity from HIV-1 was analyzed with a purified recombinant p66/p51 reverse transcriptase RT/RNase H protein and RNA-DNA hybrid consisting of RNA harboring the polypurine tract (ppt) and three complementary synthetic DNA oligonucleotides. Upon incubation of this preformed RNA-DNA hybrid with the p66/p51 RT/RNase H, a cleavage pattern is observed that indicates endonucleolytic RNase H activity with some sequence specificity for the next to last nucleotide of the 3'-end of the ppt RNA and one cut within the ppt. The RNase H avoids cleavage of G or A stretches. During RNA-directed DNA synthesis the RNA is hydrolyzed in a concerted action of RT and RNase H whereby the RNase H exhibits endonuclease as well as 3'-5'-exonuclease activity. The distance between the active centers of the RT and RNase H corresponds to 18 base pairs of the RNA-DNA hybrid. Plus-strand DNA-directed DNA synthesis initiates exactly at the next to last nucleotide of the 3'-end of the ppt RNA by means of the RNase H activity.
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PMID:Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids. 170 2

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.
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PMID:Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity. 170 63

A primase-reverse-transcriptase of Halobacterium halobium was purified by column chromatography on DEAE-cellulose, hydroxyapatite and carboxymethyl-cellulose, followed by sedimentation on a glycerol gradient. The enzyme is a multifunctional enzyme containing reverse transcriptase. DNA polymerase and RNase H activities and does not require a performed primer to initiate DNA synthesis. Using a single-stranded DNA as template, this enzyme synthesizes oligonucleotides (8-12 bases) that can be used a primer by Escherichia coli DNA nucleotidyltransferase I (DNA polymerase I, Klenow fragment). Two polypeptides of 67 and 57 kDa were found after 14750-fold purification of the enzyme.
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PMID:Reverse transcriptase in archaebacteria. Purification and characterization of a primase-reverse-transcriptase complex from Halobacterium halobium. 170 56


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