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)

We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (+)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (+)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNA-primed (-)-strand DNA synthesis.
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PMID:Analysis of efficiency and fidelity of HIV-1 (+)-strand DNA synthesis reveals a novel rate-limiting step during retroviral reverse transcription. 1109 4

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA(3)(Lys) primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5' end of the RNA that remained annealed to the 3' end of the minus-strand strong-stop DNA only if NC was present in the reaction.
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PMID:In vitro analysis of human immunodeficiency virus type 1 minus-strand strong-stop DNA synthesis and genomic RNA processing. 1113 81

UV irradiation of Escherichia coli tmRNA both on and off the ribosome induced covalent cross-links between its 3'- and its 5'-terminal segments. Cross-linking was unaffected in a molecule that lacked the tag-peptide codon region and pseudoknots 2, 3, and 4. Intact and truncated cross-linked tmRNAs were aminoacylated as efficiently as the respective nonirradiated molecules, suggesting that the added UV-induced bonds did not disturb tmRNA conformation. Using RNase H digestion followed by primer extension with reverse transcriptase, two cross-linked sites were identified within the tRNA-like region of tmRNA. The first was formed between nucleotides U9/U10 near the 5' end and nucleotides C346/U347 in the T loop. The second cross-link involved residues at positions 25-28 and 326-329 within helix 2a. Together with comparative sequence analysis, these findings yielded a three-dimensional model of the tRNA-like domain of E. coli tmRNA. Despite significant reduction of the D domain and the proximity of U9/U10 and C346/U347, the model closely resembles the L-shaped structure of canonical tRNA.
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PMID:Three-dimensional folding of the tRNA-like domain of Escherichia coli tmRNA. 1158 58

Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2'-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis were detected in the five organisms. Eight of the methylated nucleotides and one psi have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.
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PMID:Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria. 1191 66

We report the isolation and characterization of a copia-like retrotransposon, Panzee, from pigeonpea ( Cajanus cajan). The 4947-bp Panzee element is AT rich (60%) and the integrated element is flanked by a target-site duplication of 5 bp. The structure of Panzee is that of a typical LTR-retrotransposon containing long terminal repeats (LTRs) which flank its internal region. The 5' LTR is 372 bp in length and the 3' LTR is 383 bp long. Both LTRs start with 5'-TG and end with CA-3' and have 4-bp terminal inverted repeats. The internal region between the LTRs contains two priming sites for DNA synthesis: the first, a 12-bp primer binding site complementary to initiator methionyl tRNA, is located adjacent to the 3' end of the 5' LTR and the other, a 12-bp polypurine tract lies just upstream to the 5' end of the 3' LTR. The putative polyprotein shows homology to all the proteins encoded by LTR retrotransposons, i.e. group-associated antigen ( gag), proteinase, endonuclease, reverse transcriptase (RT) and ribonuclease H (RNase H). However, the cloned copy of the element contains four frameshifts and a premature stop codon in its protein-coding domain. Genomic Southern hybridization experiments using probes derived from three different regions of the element show that Panzee or Panzee-related elements are present in high copy numbers in the pigeonpea genome. Analysis of transgenic tobacco plants containing the LTR:GUS construct shows that the 5' LTR of Panzee drives gene expression in this heterologous system in a tissue-specific manner. A phylogenetic tree constructed using reverse transcriptase sequences places Panzee in the copia group of retrotransposons.
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PMID:Panzee, a copia-like retrotransposon from the grain legume, pigeonpea ( Cajanus cajan L.). 1207 29

A photoreactive analogue of spermine, N1-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5' domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem-loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.
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PMID:The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions. 1208 67

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.
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PMID:Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo. 1209 8

None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.
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PMID:Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format. 1218 96

Successful long-term management of HIV infection will require targeted inhibition of multiple steps essential for virus replication. Currently, both nucleoside- and nonnucleoside-based inhibitors of DNA polymerase function, in combination with antagonists of HIV protease, have been shown to be clinically beneficial. However, it is clear that RNase H activity of the multifunctional HIV-1 reverse transcriptase (RT) is absolutely required for completion of retroviral DNA synthesis, thereby rendering this function an attractive target for drug development. Although generally viewed as a sequence-independent activity, highly precise RNase H cleavage is required in order to remove the RNA primers of (-) and (+) strand DNA synthesis (a host-derived tRNA and the polypurine tract, respectively), thereby preserving the ends of linear DNA and facilitating integration. The availability of highly purified, recombinant RT/RNase H has allowed a thorough dissection of these multiple events and their potential for therapeutic intervention. Our current understanding of retroviral RNase H function and the status of small molecule inhibitors are the focus of this review.
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PMID:Uncovering the complexities of retroviral ribonuclease H reveals its potential as a therapeutic target. 1255 93

Taro bacilliform virus (TaBV) has been classified as a putative badnavirus based on its non-enveloped, bacilliform virion morphology and transmission by mealybugs. The complete nucleotide sequence of a Papua New Guinea isolate of TaBV has now been determined and comprises 7458 bp. The genome contains four open reading frames (ORFs) on the plus-strand that potentially encode proteins of 17, 16, 214 and 13 kDa. The size and organisation of TaBV ORFs 1-3 is similar to that of most other badnaviruses, while the location of ORF 4 is similar to that of ORF 4 and ORF X of the atypical badnaviruses Citrus yellow mosaic virus and Cacao swollen shoot virus, respectively. The putative amino acid sequence of TaBV ORF 3 contained motifs that are conserved amongst badnavirus proteins including aspartic protease, reverse transcriptase (RT) and ribonuclease H (RNase H). The highly conserved putative plant tRNA(met)-binding site was also present in the 935 bp intergenic region of TaBV. Phylogenetic analysis using the amino acid sequence of ORF 3 showed that TaBV branched most closely to Dioscorea bacilliform virus. These results confirm that TaBV is a pararetrovirus of the genus Badnavirus, family Caulimoviridae.
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PMID:Genomic characterisation of taro bacilliform virus. 1272 1


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