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)

Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli. The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279. The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69. The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E. coli RNase HI, which shows 52% amino acid sequence identity. Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues. The enzymatic activities of T. thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E. coli RNase HI at 37 degrees C. The melting temperature, Tm, of T. thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E. coli RNase HI. The free energy changes of unfolding in the absence of denaturant, delta G[H2O], of T. thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E. coli RNase HI.
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PMID:Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8. 131 54

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.
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PMID:Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart. 132 37

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.
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PMID:Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli. 164 58

To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.
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PMID:Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond. 184 45

A double stranded RNA species has been detected in guanidine hydrochloride extracts of mitochondria from respiratory competent cells of Saccharomyces cerevisiae. This novel mitochondrial RNA, termed mtdsRNA, has been purified in a Cs2SO4 density gradient where it bands at a density of 1.58 g/ml. The mtdsRNA runs as a single slow moving band on agarose gels. Its double stranded RNA character was evidenced by its sensitivity to digestion by RNase III, but not by RNase H, or DNase I. Moreover the mtdsRNA hybridized to each separated strand of a petite mtDNA. It is concluded that mtdsRNA contains long transcripts derived from most regions of yeast mtDNA, because 1) its weight-average length as determined by electron microscopy was 4.5 micrometer (about 14 kb, or 20% of the wild type mtDNA genome), and 2) it hybridized to each of a series of eight petite mtDNA probes carrying sequences derived from widely different segments of mtDNA. It is proposed that prolonged transcription of both strands of yeast mtDNA can occur and that mtdsRNA arises from hybridization of these long complementary transcripts.
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PMID:A novel species of double stranded RNA in mitochondria of Saccharomyces cerevisiae. 627 33

A genetic method for isolating a mutant enzyme of ribonuclease HI (RNase HI) from Thermus thermophilus HB8 with enhanced activity at moderate temperatures was developed. T. thermophilus RNase HI has an ability to complement the RNase H-dependent temperature-sensitive (ts) growth phenotype of Escherichia coli MIC3001. However, this complementation ability was greatly reduced by replacing Asp(134), which is one of the active site residues, with His, probably due to a reduction in the catalytic activity. Random mutagenesis of the gene encoding the resultant D134H enzyme, followed by screening for second-site revertants, allowed us to isolate three single mutations (Ala(12) --> Ser, Lys(75) --> Met, and Ala(77) --> Pro) that restore the normal complementation ability to the D134H enzyme. These mutations were individually or simultaneously introduced into the wild-type enzyme, and the kinetic parameters of the resultant mutant enzymes for the hydrolysis of a DNA-RNA-DNA/DNA substrate were determined at 30 degrees C. Each mutation increased the k(cat)/K(m) value of the wild-type enzyme by 2.1-4.8-fold. The effects of the mutations on the enzymatic activity were roughly cumulative, and the combination of these three mutations increased the k(cat)/K(m) value of the wild-type enzyme by 40-fold (5.5-fold in k(cat)). Measurement of thermal stability of the mutant enzymes with circular dichroism spectroscopy in the presence of 1 M guanidine hydrochloride and 1 mM dithiothreitol showed that the T(m) value of the triple mutant enzyme, in which all three mutations were combined, was comparable to that of the wild-type enzyme (75.0 vs 77.4 degrees C). These results demonstrate that the activity of a thermophilic enzyme can be improved without a cost of protein stability.
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PMID:Enhancement of the enzymatic activity of ribonuclease HI from Thermus thermophilus HB8 with a suppressor mutation method. 1105 82

Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.
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PMID:Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA). 1521 74

Inhibitor resistance of several commercial Moloney murine leukemia virus reverse transcriptase (MMLV RT) enzymes was investigated. IC(50) values were determined for potential RNA contaminants, including guanidine thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related acidic polysaccharides. Sensitivity (as judged by MMLV RT IC(50) values) was directly correlated to the outcome of "mock" reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays carried out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sensitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RT pentuple mutant (E69K/E302R/W313F/L435G/N454K) showed higher tolerance to these substances than the wild type. Increased resistance was also noted in RT-qPCR comparisons employing crude cell lysates.
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PMID:Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors. 2010 Apr 52

The properties of gapmer antisense oligonucleotide (ASO) flanked by deoxyribonucleic guanidine (DNG) were investigated for the potential application in antisense technology. DNG is a unique nucleotide analog which has a positively charged internucleotide guanidinium linkage instead of negatively charged phosphodiester backbone linkage. We prepared a gapmer ASO containing DNG units at both wings of the sequence and compared its properties with 2',4'-BNA/LNA gapmer ASOs with phosphorothioate (PS) backbone. Although DNG gapmer showed no stabilizing effect on the duplex formation with target RNA, the DNG modification was found to be tolerant to exonuclease digestion. Furthermore, DNG gapmer can induce RNase H-mediated cleavage of target RNA molecule, a requisite property for the antisense strategy. Therefore, the DNG gapmer developed in this study could be an interesting and useful candidate for the development of potent ASOs.
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PMID:Development of gapmer antisense oligonucleotide with deoxyribonucleic guanidine (DNG) modifications. 3155 56

The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2'-O-methylribonucleotides (2'-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2'-OMe PGO in mycobacteria. The results indicated that 2'-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2'-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2'-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2'-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2'-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.
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PMID:A New Antisense Phosphoryl Guanidine Oligo-2'-O-Methylribonucleotide Penetrates Into Intracellular Mycobacteria and Suppresses Target Gene Expression. 3163 66


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