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)

Illimaquinone, a natural marine product, was shown by us to inhibit preferentially the ribonuclease H (RNase H) activity of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). We have also shown that illimaquinone inhibits the RNase H activity of HIV-2 RT in addition to that of HIV-1 RT, murine leukemia virus RT, and Escherichia coli RNase H. Chemical modifications of HIV-1 RT by sulfhydryl-specific reagents, such as N-ethylmaleimide (NEM) have been demonstrated to specifically inhibit the RNase H activity of the enzyme. Since our previous studies have suggested that cysteine 280 in HIV-1 RT interacts with the sulfhydryl reagents, we have examined the possibility that illimaquinone interacts with the RT molecules via amino acid residues located in the vicinity of cysteine 280 in both HIV-1 and HIV-2 RTs. In the combined effect studies of illimaquinone and NEM, the two structurally unrelated compounds were shown to be mutually exclusive, exhibiting an antagonistic interaction with both HIV-1 and murine leukemia virus-associated RNase H activities. This implicates cysteine 280, in both HIV-1 and HIV-2 RTs, to be in close proximity to the putative binding site of the enzyme to illimaquinone. The above conclusion is further supported by the fact that the RNase H activity of an enzymatically active mutant of HIV-1 RT, in which cysteine 280 was replaced by serine, was substantially more resistant to illimaquinone than the corresponding activity of the wild-type enzyme. The fact that NEM failed to inhibit E. coli RNase H as opposed to illimaquinone highlights a major difference between the retroviral and bacterial RNase H.
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PMID:The interaction of illimaquinone, a selective inhibitor of the RNase H activity, with the reverse transcriptases of human immunodeficiency and murine leukemia retroviruses. 768 48

On the basis of the reported enhanced antisense activity of polylysine-oligonucleotide conjugates, a synthetic 12-mer oligodeoxyribonucleotide has been coupled at its 5' terminus to a series of positively charged (delta-ornithine)n cysteine peptides. Binding between the nucleic acid-peptide conjugate and its complementary DNA target sequence was detected by the impact of complexation on the melting temperature (Tm). It was found that the Tm for the nucleic acid-peptide gradually increased with increasing net charge on the conjugated peptide. Site-directed cleavage with RNase H demonstrates that the peptide-modified oligomer also hybridizes with its RNA target sequence. Increased affinity for target mRNA with net charge was shown by a cell-free translation arrest assay.
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PMID:Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA. 828 27

A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotides (ODNs) to determine which are most effective for mRNA translation-arrest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of the T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under test. Transcription and translation were accomplished in a one-tube reaction. Translation-arrest caused by antisense ODN was evaluated in terms of the amounts of de novo-synthesized, [35S]-methionine or [35S]cysteine labeled target protein measured by gel electrophoresis and autoradiography. The properties of this system and optimal reaction conditions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.
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PMID:A method for screening antisense oligodeoxyribonucleotides effective for mRNA translation-arrest. 888 14

Treatment of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with N-ethylmaleimide (NEM) selectively inhibits the RNase H activity. The cysteine residue at position 280 (C280) is the target for NEM; HIV-1 RT carrying the mutation C280S is resistant to NEM. Since HIV-1 RT is composed of two related subunits (p66 and p51) that play distinct roles, we asked whether the C280 in p51 or the C280 in p66 is responsible for the sensitivity of the enzyme to NEM. HIV-1 RT versions were prepared that had one mutant and one wild-type subunit. When these chimeric enzymes were tested, both the p51 and p66 subunits were found to contribute to the sensitivity of the enzyme to NEM. The implications of these results are discussed in the context of the structure of the enzyme.
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PMID:Subunit-specific mutagenesis of the cysteine 280 residue of the reverse transcriptase of human immunodeficiency virus type 1: effects on sensitivity to a specific inhibitor of the RNase H activity. 918 46

Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.
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PMID:Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. 1033 Jan 72

Stability parameters for individual residues in Thermus thermophilus cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that T. thermophilus RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.
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PMID:Structural distribution of stability in a thermophilic enzyme. 1057 Jan 31

Cys(38) and Cys(280) of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross-linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C(561)) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21. Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies.
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PMID:Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking. 1074 61

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in alpha helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the K(m) rather than changes in the k(cat) values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA-DNA substrate.
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PMID:Mutagenesis of cysteine 280 of the reverse transcriptase of human immunodeficiency virus type-1: the effects on the ribonuclease H activity. 1261 5

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5' hydroxyl groups as well as 3' hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.
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PMID:Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay. 1708 78

The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI.
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PMID:Crystal structure of type 1 ribonuclease H from hyperthermophilic archaeon Sulfolobus tokodaii: role of arginine 118 and C-terminal anchoring. 1789 5


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