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)

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.
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PMID:Investigating the structure of human RNase H1 by site-directed mutagenesis. 1131 19

The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function.
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PMID:Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site. 1134 99

The natural form of the human immunodeficiency virus type one reverse transcriptase (HIV-1 RT) found in virion particles is a heterodimer composed of the p66 and p51 subunits. The catalytic activity resides in the larger subunit in the heterodimeric (p66/p51) enzyme while in the monomeric form it is inactive. In contrast, Murine leukemia virus RT (MuLV RT) is functionally active in the monomeric form. In the primary amino acid sequence alignment of MuLV RT and HIV-1 RT, we have identified three specific regions in MuLV RT, that were missing in HIV-1 RT. In a separate study, we have shown that a chimeric RT construct comprising of the polymerase domain of HIV-1 RT and RNase-H domain of MuLV RT is functionally active as monomer [20]. In this communication, we demonstrate that insertion of a peptide (corresponding to amino acid residues 480-506) from the connection subdomain of MuLV RT into the connection subdomain of HIV-1 RT (between residues 429 and 430) results in a functionally active monomeric chimeric RT. Furthermore, this chimeric enzyme does not dimerize with exogenously added p51 subunit of HIV-1RT. Functional analysis of the chimeric RT revealed template specific variations in its catalytic activity. The chimeric enzyme catalyzes DNA synthesis on both heteropolymeric DNA and homopolymeric RNA (poly rA) template but curiously lacks reverse transcriptase ability on heteropolymeric RNA template. Similar to MuLV RT, the polymerase activity of the chimeric enzyme is not affected by acetonitrile, a reagent which dissociates dimeric HIV-1 RT into inactive monomers. These results together with a proposed 3-D molecular model of the chimeric enzyme suggests that the insertion of the missing region may induce a change in the spatial position of RNase H domain such that it is functionally active in monomeric conformation.
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PMID:Insertion of a peptide from MuLV RT into the connection subdomain of HIV-1 RT results in a functionally active chimeric enzyme in monomeric conformation. 1171 55

Escherichia coli RNase HI is a well-characterized model system for protein folding and stability. Controlling protein stability is critical for both natural proteins and for the development of engineered proteins that function under extreme conditions. We have used native-state hydrogen exchange on a variant containing the stabilizing mutation Asp10 to alanine in order to determine its residue-specific stabilities. On average, the DeltaG(unf) value for each residue was increased by 2-3 kcal/mol, resulting in a lower relative population of partially unfolded forms. Though increased in stability by a uniform factor, D10A shows a distribution of stabilities in its secondary structural units that is similar to that of E. coli RNase H, but not the closely related protein from Thermus thermophilus. Hence, the simple mutation used to stabilize the enzyme does not recreate the balance of conformational flexibility evolved in the thermophilic protein.
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PMID:Native-state energetics of a thermostabilized variant of ribonuclease HI. 1173 3

We have analyzed the response of a number of human cell lines to treatment with antisense oligodeoxynucleotides (ODNs) directed against RNA polymerase II, replication protein A, and Ha-ras. ODN-delivery to the cells was liposome-mediated or via electroporation, which resulted in different intracellular locations of the ODNs. The ODN-mediated target mRNA reduction varied considerably between the cell lines. In view of the essential role of RNase H activity in this response, RNase H was analyzed. The mRNA levels of RNase H1 and RNase H2 varied considerably in the cell lines examined in this study. The intracellular localization of the enzymes, assayed by green-fluorescent protein fusions, showed that RNase H1 was present throughout the whole cell for all cell types analyzed, whereas RNase H2 was restricted to the nucleus in all cells except the prostate cancer line 15PC3 that expressed the protein throughout the cell. Whole cell extracts of the cell lines yielded similar RNase H cleavage activity in an in vitro liquid assay, in contrast to the efficacy of the ODNs in vivo. Overexpression of RNase H2 did not affect the response to ODNs in vivo. Our data imply that in vivo RNase H activity is not only due to the activity assayed in vitro, but also to an intrinsic property of the cells. RNase H1 is not likely to be a major player in the antisense ODN-mediated degradation of target mRNAs. RNase H2 is involved in the activity assayed in vitro. The presence of cell-type specific factors affecting the activity and localization of RNase H2 is strongly suggested.
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PMID:The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides. 1185 17

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

Oligonucleotide-targeted RNase H protection assays are powerful means to analyze protein binding domains in ribonucleoprotein particles (RNPs). In such an assay, the RNA component of a RNP and, in an essential control reaction, the corresponding deproteinized RNA are targeted with an antisense DNA oligonucleotide and RNase H. If the oligonucleotide is able to anneal to the complementary sequence of the RNA, RNase H will cleave the RNA within the double-stranded DNA/RNA region. However, protein binding to a specific RNA sequence may prevent hybridization of the DNA oligonucleotide, thereby protecting the RNA molecule from endonucleolytic cleavage. An RNase H protection analysis can usually be carried out with crude cell extract and does not require further RNP purification. On the other hand, purified RNP fractions are preferable when a crude extract contains RNase activity or a heterogenous RNP population of a specific RNA. The cleavage pattern of RNase H digestion can be analyzed by Northern blotting or primer-extension assays. In addition, the investigation of RNP fragments, for example, by native gel electrophoresis, may reveal important structural information about a RNP. In this article, we describe procedures for RNP and RNA preparation, the oligonucleotide-targeted RNase H protection assay, and methods for the analysis of RNA and RNP cleavage products. As an example, we show oligonucleotide-targeted RNase H protection of the Trypanosoma brucei U1 small nuclear RNP.
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PMID:Analysis of RNA-protein complexes by oligonucleotide-targeted RNase H digestion. 1205 93

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.
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PMID:Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H. 1218 80

A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA. Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined. To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA. These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790. Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA. Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.
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PMID:Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H. 1236 83

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.
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PMID:Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome. 1241


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