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:3.1.27.1 (RNase)
16,360 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

The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.
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PMID:Structural and functional analysis of the visna virus Rev-response element. 131 70

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.
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PMID:Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. 137 Oct 14

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

We have used transient expression assays to study transcription directed by the human immunodeficiency virus (HIV) type 1 promoter. A plasmid containing an HIV-reporter gene fusion and a simian virus 40 origin of DNA replication was transfected into COS-1 cells in the presence or absence of a Tat expression vector. HIV-promoted RNA was analyzed by in vivo labeling, by RNase protection mapping, and in run-on transcription assays. As observed previously, two populations of HIV RNA accumulate in vivo: short, attenuated transcripts and long, polyadenylated mRNA. The short transcripts labeled in vivo were longer and more heterogeneous than expected from RNase protection assays. Moreover, comparison of transcripts labeled in vivo with run-on transcription products revealed that similar, if not identical, short RNAs accumulate in vitro. Utilizing the run-on assay, we show that following transcriptional termination, the attenuated transcripts undergo processing to generate one species of RNA. We also provide evidence that Tat does not act as an antiterminator to relieve a discrete elongation block but instead modifies transcriptional complexes, enabling them to overcome putative pause sites and continue transcription of the template.
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PMID:Premature termination and processing of human immunodeficiency virus type 1-promoted transcripts. 160 55

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

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
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PMID:Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). 168 23

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.
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PMID:Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase. 169 Oct 20

HIV (human immunodeficiency virus) viraemia in serum or plasma of HIV-infected individuals was investigated by the polymerase chain reaction assay (PCR) in combination with reverse transcription to detect HIV-1 genomic RNA. Before PCR, plasma or serum was ultracentrifuged, precipitated virions were then treated with a RNase-free DNase, and a cDNA from the HIV-1 genomic RNA was synthesized. Thirty-three fresh plasma and seven sera from either HIV-1 antibody-positive individuals or patients treated with AZT were tested. Plasma from three patients were assayed 3 or 6 months apart. Twelve sera from HIV-1 antibody-negative individuals were used as negative control. PCR was performed with primers in LTR, gag and env regions: 11 of 40 samples were positive with three primer pairs, 16 with two primer pairs and 11 with only one primer pair. PCR on HIV-1 genomic cDNA was positive in 38 out of the 40 plasma or serum samples (95%), regardless of the clinical stage of the infection: HIV-1 was detected in 14 of the 15 untreated subjects and in 24 of the 25 AZT-treated patients. HIV p24 antigen was detected in the serum of 38% of subjects (15 of 40). The results suggest that this method is suitable for the detection of viral particles in plasma or serum from HIV-1-infected individuals irrespective of antiviral treatment.
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PMID:The polymerase chain reaction for the detection of HIV-1 genomic RNA in plasma from infected individuals. 171 16


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