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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.
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PMID:Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. 137 66

Reverse transcriptase sequences, which are fundamental to retrovirus existence, are widely distributed in the living world. Phylogenies based on their sequences set vertebrate retroviruses apart as relatively modern creations. Their nearest evolutionary relatives are a large group of transposable elements that have all the standard retrovirus equipment except spliced envelope proteins. The distribution of these elements suggests a long-standing presence predating the radiation of plants, fungi, and animals. There is another large group of elements, LINEs, that also contain recognizable reverse transcriptase sequences and which likely diverged even earlier, as evidenced by their presence in trypanosomes and other protists. They lack tRNA priming sites--which they could have lost--but they do exhibit characteristic eukaryotic polyadenylation. These elements are problematic in that the sequences are so degenerate in most instances that it is not possible to identify the accessory enzymes or structural proteins with any confidence, leaving major gaps in our reconstruction of events. Even with these gaps, however, the historical beginnings of retroviruses can be traced back to events coincident with the prokaryotic invasion of primitive eukaryotes.
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PMID:Tracing the origin of retroviruses. 137 25

The kinetics of copying of poly(A).(dT)n, poly(A).(U)n, poly(dA).(dT)n and poly(A).(dT)9-U by reverse transcriptase of human immunodeficiency virus-1 (HIV-1) has been studied and the binding affinity of the enzyme, for template or primer, determined. Short oligonucleotides and dTTP served as primers in the HIV-1 reverse-transcriptase-dependent DNA synthesis. Km and Vmax were measured as functions of the primer chain length; the logarithm of the values of both Km and Vmax increased linearly up to 10. For longer primers (n = 11 to n = 24) the increase of those values changes very little. The enhanced affinity of the primers, (dT)n or (U)n due to the formation of one complementary pair, A.dT, dA.dT, A.U was estimated as a factor of 2. A specific property of HIV-1 reverse transcriptase compared with other DNA polymerases (procaryotes, eucaryotes, other retroviruses and archaebacteria) was its higher affinity to riboprimers as compared to deoxyriboprimers. Relative initial rates when copying poly(A) or poly(dA) templates using different primers and various conditions were compared; the optimal temperature for the reaction of polymerization with poly(A) or poly(dA) templates and (U)10, (dT)10 or (dT)9-U primers was determined. The maximal activity of the enzyme in the case of poly(A) and decanucleotide primers was found at temperatures between 27-31 degrees C. An increase in the primer length results in the stabilization of the template.primer duplex complexed to the enzyme, thus increasing to more than 40 degrees C the optimal temperature of polymerization. The activation energy (Ea) values of the polymerization reaction for different template.primer complexes were evaluated.
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PMID:Functional analysis of primers and templates in the synthesis of DNA catalyzed by human immunodeficiency virus type 1 reverse transcriptase. 137 4

Studies from several laboratories have provided evidence that distinct stromal cell-derived signals are involved in the maturation of pre-B cells into surface Ig expressing B lymphocytes. In order to define the stage of development at which these stimuli act, various polymerase chain reaction strategies were used to characterize the status of kappa L chain gene rearrangements in nontransformed, stromal cell dependent pre-B cells. These cells were obtained from lymphoid colonies whose growth was potentiated by factors from a stromal cell line. kappa L chain genes in cells from many of these colonies were rearranged, and analysis of the Jk genes used indicated a bias toward the most 3' loci. However, the use of a reverse transcriptase PCR strategy failed to detect mature kappa transcripts, indicating that stromal cell mediators exist that allow pre-B cells to progress to the stage at which L chain genes are rearranged but not expressed. Reverse transcriptase PCR further revealed that no transcripts for c-kit (the receptor for kit-ligand) and the IL-7R could be detected in these cells. This suggests that these receptors are no longer expressed by the time cells have undergone kappa rearrangements and minimize a role for stromal cell-derived kit-ligand and IL-7 in mediating the pre-B to B cell transition.
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PMID:Status of kappa L chain gene rearrangements and c-kit and IL-7 receptor expression in stromal cell-dependent pre-B cells. 138 91

Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed.
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PMID:Regulation of the reverse transcriptase of human immunodeficiency virus type 1 by dNTPs. 138 60

Although the origin of viruses has not yet been clarified, definite differences in evolutionary patterns have been found among RNA. Retro and DNA viruses. These differences are reflected in infectious diseases. RNA viruses, which have RNA in their genome, replicate many times over in cells within a short period of time, destroying the host cells and causing an acute infection. As the error frequency of RNA replicase enzymes is high, the rate of evolution of RNA viruses is very rapid. Retroviruses also contain RNA as their genome, but the genome RNA is reversely transcribed to the DNA in nuclei and then incorporated into the host chromosome to replicate. The error frequency of reverse transcriptase is also high, and therefore mutations easily occur as well. The transcribed DNA is integrated into host DNA in the nucleus, and it remains in the integrated state for human entire life time, causing chronic disease or developing malignant tumors. As DNA viruses except poxviruses replicate inside the cell nucleus and the error frequency of DNA polymerase is low, the speed of mutation and the degree of resulting diversity are lower than those in the case of the RNA virus. DNA viruses tend to stay inside the body for long periods of time and easily become latent. In this paper, I shall discuss 1) the nature of viruses, 2) the origin of viruses, 3) mutation and recombination, 4) diversity of RNA viruses, 5) quickly changing viral diseases, 6) eradicated viral diseases, 7) chronic and malignant diseases, and 8) control of viral diseases.
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PMID:[Evolution and ecological changes of animal viruses]. 140 23

Several antitumor substances that effectively inhibited the growth of ascites and solid tumor cells transplanted in mice were isolated from pine cone NaOH extract by acid- and ethanol-precipitation. These antitumor substances were also potent antiviral agents against human immunodeficiency virus, herpes simplex virus and influenza virus; they induced antimicrobial activity against Staphylococcal aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, and induced antiparasite activity against Hymenolepis nana in mice. Chemical analysis of these substances by IR, UV, NMR, ESR and partition chromatography on cellulose-TLC plate disclosed that they had lignin-related structures complexed with sugars or polysaccharides. Chlorinated decomposition of the lignin portion significantly reduced their antiviral activity. In agreement with this, the antiviral activity of synthesized lignins prepared by polymerization of phenylpropanoid precursors was comparable to that of the undecomposed counterparts of the pine cone extract. Acid hydrolysis of the polysaccharide portion significantly reduced the ability of the substances to induce antitumor and antimicrobial activities in mice. With an appropriate eliciting agent, intravenous administration of natural lignified substances transiently induced endogenous production of a cytotoxic factor (possibly tumor necrosis factor) in normal mice. Their priming activity was significantly higher than that of their component units or degradation products. These data suggest the importance of conjugating lignins with polysaccharides for in vivo expression of various kinds of immunopotentiating activity. As possible explanations for their induction of a variety of immunopotentiating activities, these natural and synthetic lignins stimulated macrophage NBT-reducing activity, polymorphonuclear cell (PMN) iodination and splenocyte DNA synthesis and inhibited poly (ADP-ribose) glycohydrolase, RNA-dependent DNA polymerase (reverse transcriptase) and RNA-dependent RNA polymerase activities.
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PMID:Antitumor, antiviral and immunopotentiating activities of pine cone extracts: potential medicinal efficacy of natural and synthetic lignin-related materials (review). 164 35

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
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PMID:Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. 168 98

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
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PMID:Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. 168 52

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.
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PMID:Characterization of reverse transcriptase from feline immunodeficiency virus. 169 Jul 35


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