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

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

Because of their nuclease resistance and ability to form substrates for RNase H, antisense oligodeoxynucleotides (ODNs) possessing several methoxyethylphosphoramidate linkages at both termini have proven effective at targeting the degradation of specific mRNAs in Xenopus embryos. The efficacy of these compounds subsequently observed in tissue culture focused our attention on the issue of cellular uptake. To investigate the extent to which phosphate backbone modifications may increase the lipophilicity of ODNs, and thereby increase passive uptake by cells, the partitioning of a series of phosphoramidate-modified compounds between aqueous and organic phases was examined. The octanol:water partition coefficient of an unmodified, mixed-sequence 16-mer was 1.75 x 10(-5). The log of the partition coefficient increased in a sigmoidal manner with the number of methoxyethylphosphoramidate internucleoside linkages, indicating a nonlinear free energy relationship. The highest level of partitioning demonstrated was approximately 4 x 10(-3) (a 230-fold increase), attained when 11 of the 15 phosphodiesters were modified. An increase in hydrophobicity was also attained with C8 and C10 alkylamines acting as phase-transfer agents. The melting temperatures of heteroduplexes formed between a phosphoramidate-modified ODN and a complementary unmodified DNA strand decreased by approximately 1.5 degrees C for every phosphate group modification. ODNs can thus be extensively derivatized without substantially compromising duplex formation under physiological conditions.
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PMID:Physical properties of oligonucleotides containing phosphoramidate-modified internucleoside linkages. 203 Sep 62

Earlier studies had shown that a large portion of bacterial messenger RNA carries 3'-terminal polyadenylate sequences, albeit of somewhat shorter length than those associated with eukaryotic mRNA. In this paper, we show for the first time that a specific prokaryotic mRNA is polyadenylated. Three independent lines of evidence demonstrate that a 3'-terminal polyadenylate sequence 10 to 15 nucleotides in length is associated with about 40% of the mRNA of the outer membrane lipoprotein of Escherichia coli: 40% of lipoprotein mRNA binds to oligodeoxythymidylate-substituted cellulose at high ionic strength and is eluted by water; treatment of lipoprotein mRNA with oligodeoxythymidylate and ribonuclease H destroys its ability to bind to oligodeoxythymidylate-cellulose; and in the presence of oligodeoxythymidylate, lipoprotein mRNA can serve as template for the synthesis of DNA complementary to lipoprotein mRNA by reverse transcriptase. In view of the fact that the lpp gene and its downstream-flanking region contain no continuous deoxyadenylate sequences longer than five nucleotides, the polyadenylate moiety must be added post-transcriptionally. It was possible to demonstrate the synthesis of polyadenylated lipoprotein mRNA in toluene-permeabilized cells of E. coli, opening the way for the study of its biosynthesis.
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PMID:Messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane is polyadenylated. 243 23

Our earlier studies have shown that the mRNA from many bacterial species, including Escherichia coli and Bacillus subtilis, is extensively polyadenylated, but with shorter poly(A) segments than those associated with eukaryotic mRNA. In this paper, we show that about 40% of the mRNA for the tryptophan synthetase alpha-subunit (TrpA) of E. coli carries a 3'-terminal polyadenylate sequence of 15 to 20 residues. This conclusion was supported by several independent lines of evidence. About 40% of trpA mRNA bound to oligo(dT)-cellulose at high ionic strength and was eluted with water. Treatment with RNase H in the presence of oligo(dT)12-18 destroyed the ability of trpA mRNA to bind to oligo(dT)-cellulose, presumably through the degradation of the poly(A) tract. trpA mRNA could be used as template for complementary DNA synthesis with reverse transcriptase in a reaction that was absolutely dependent on oligo(dT)12-18 as primer. The identity of the cDNA product as a complement to trpA mRNA was established by specific hybridization. In addition, it was possible to synthesize polyadenylated trpA mRNA in toluene-permeabilized cells of E. coli transformed with a recombinant plasmid carrying the trpA gene. In view of the fact that the trpA gene and its 3'-untranslated region contain no continuous deoxyadenylate sequences larger than five nucleotides, one can conclude that the polyadenylate moiety is added post-transcriptionally.
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PMID:3'-terminal polyadenylate sequences of Escherichia coli tryptophan synthetase alpha-subunit messenger RNA. 244 21

The backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) in the picosecond to nanosecond time scale were characterized by a combination of measurements of 15N-NMR relaxation (T1, T2, and NOE), analyzed by a model-free approach, and molecular dynamics (MD) simulation in water. The MD simulations in water were carried out with long-range Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the 15N-NMR relaxation indicated that RNase HI has a rotational correlation time of 10.9 ns at 27 degrees C. The generalized order parameter (S2) for the internal motions varied from 0.15 to 1.0, with an average value of 0.85, which is much larger than that of the RNase H domain of HIV-1 reverse transcriptase (0.78). Large internal motions (small order parameters) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I and beta-strand D (Glu61, His62), the loop between beta-strand D and alpha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV (Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123-His127), and the C-terminal region (Gln152-Val155). The effective correlation time observed in these regions varied from 0.45 ns (Glu61, Lys96) to 2.2 ns (Leu14). The order parameters calculated from the MD agreed well with those from the NMR experiment, with a few exceptions. The distributions of most of the backbone N-H vectors obtained by MD are approximately consistent with the diffusion-in-a-cone model. These distributions, however, were elliptic, with a long axis perpendicular to the plane defined by the N-H and N-C alpha vectors. Distributions supporting the axial fluctuation model or the jump-between-two-cones model were also observed in the MD simulation.
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PMID:Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination of dynamic models. 775 90

Few inhibitors of the RNase H function associated with the HIV-1 reverse transcriptase have been discovered to date. We observed that three novenamines, U-34445, U-35122, and U-35401, are specific inhibitors of the HIV-1 RT RNase H function. All three compounds are strong amphiphiles and contain one ionizable group. Hence, a priori, in aqueous solutions the inhibitors might exist in at least four different physical states, namely protonated monomers, ionized monomers, protonated micelles, and ionized micelles. The three inhibitors all yielded anomalous dose-response curves, indicating that the four molecular species have different inhibitory potentials. In order to identify the inhibitory species, the amphiphilic properties of these compounds were studied. It was established that in alkaline solutions, around pH 8, all compounds are ionized and form micelles at concentrations above their CMC. Both the protonated and the ionized forms of these molecules form stable insoluble monomolecular layers at the air/water interface. The anomalies of the dose-response curves can be resolved by taking into account the fact that, in solution, the relative proportion of these molecules in each physical state depends on the pH and on their analytical concentration. Thus interpreted, the results indicate that RNase H is inhibited only by the ionized micellar form of these compounds and not by their monomeric form. Around their pKa (approximately pH 5), the three compounds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system.
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PMID:The amphiphilic properties of novenamines determine their activity as inhibitors of HIV-1 RNase H. 862 Sep 35

The effects of water deprivation on the secretion of vasotocin (AVT) and expression of the AVT gene were studied in White Leghorn cockerels. Animals deprived of water for 4 days were compared with normally hydrated controls. Blood samples were obtained for measurements of plasma osmolality and AVT levels, and the hypothalamus was collected for extraction of total cellular RNA. A 519-bp AVT cDNA was prepared by reverse transcriptase-polymerase chain reaction and a 209-bp PstI/EcoRI restriction fragment from the 3' region of the fowl AVT cDNA was used as a probe for Northern blot analysis. Plasma osmolality and AVT levels in dehydrated birds were about 30 and 350% greater, respectively, than those in normally hydrated controls. The quantity of hypothalamic AVT mRNA was 2. 3-fold greater in water-deprived birds compared to controls. The size of the hypothalamic AVT transcript was about 100-bp longer in the water-deprived birds. As determined by RNase H treatment in the presence and absence of oligo(dT)12-18, the increase in mean size of the AVT mRNA in dehydrated animals was due to a longer poly(A) tract. Our results indicate that osmotic stress up-regulates expression of the AVT gene and increases the accumulation of AVT mRNA in the hypothalamus. This accumulation may, in part, be due to lengthening of the AVT mRNA poly(A) tail which is a general mechanism associated with stabilization of vertebrate mRNAs.
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PMID:Changes in poly(A) tail length of arginine vasotocin messenger ribonucleic acid in the hypothalamus of water-deprived chickens. 881 2

Antisense experiments are often complicated by the lack of reliable methods for selecting effective antisense sequences. Chimeric oligodeoxynucleotide (ODN) libraries and ribonuclease H (RNase H) were used to identify regions on the 1253 nucleotide angiotensin type-1 receptor (AT1) mRNA that are accessible to hybridization with antisense ODNs. Phosphorothioate antisense ODNs targeted against accessible sites reduced AT1 receptor levels by at least 50% in cell culture. ODNs to 4 sites produced a 70% to 80% reduction. In contrast, most sequences targeted between accessible sites were ineffective. When injected into the brains of rats, ODNs targeted to accessible sites reduced AT1 (by 65%) but not AT2 receptor levels. Additionally, AT1 receptor function as measured by agonist-induced water intake, was significantly attenuated in these rats. ODNs directed between accessible sites were ineffective at suppressing water intake. RNA mapping can be applied to any RNA target to facilitate selection of multiple, active antisense sequences for cell culture and in vivo experiments.
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PMID:Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries. 944 87


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