Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
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PMID:Mechanism and fidelity of HIV reverse transcriptase. 128 79

The ability of peptides coded by the anti-sense strand of DNA to interact specifically with peptides coded by the sense strand has been evaluated. The sense peptide examined, ribonuclease S-peptide, was immobilized on a coated silica affinity chromatographic matrix. Anti-sense peptides were synthesized on the basis of the anti-sense DNA sequence for the S-peptide region in native pancreatic ribonuclease A. The interaction of synthetic anti-sense peptides with sense peptide was quantitated from the degree of retardation during chromatographic elution on the sense peptide affinity matrix in buffers with and without soluble competing sense peptide. Sense/anti-sense peptide interactions were found to occur with significant affinities with each of two anti-sense 20-residue peptides of opposite amino-to-carboxyl orientations and to weaken progressively with decreasing length of anti-sense peptide. The substantial chromatographic retardation of anti-sense peptides was specific, since it decreased as expected with increasing concentration of the soluble competing S-peptide, could not be mimicked by the elution of several control peptides (including S-peptide itself) on the S-peptide matrix, and did not occur with a blank chromatographic matrix (no S-peptide attached). The stoichiometry of anti-sense peptide binding to immobilized sense peptide was found to be far greater than 1:1, and at least 4-5:1, for the two 20-mer anti-sense peptides. In sum, the analytical affinity chromatographic experiments have established quantitatively that anti-sense peptide binding to sense peptides occurs in the ribonuclease S-peptide case and have identified some structural elements that govern these interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Anti-sense peptide recognition of sense peptides: direct quantitative characterization with the ribonuclease S-peptide system using analytical high-performance affinity chromatography. 356 39

A sequence-selective artificial ribonuclease was prepared by attaching ethylenediamine to the 5'-end of a DNA oligomer as the sequence-recognizing moiety. The hybrid, incorporating a 19-mer DNA which is complementary with the A44-A62 sequence of tRNA(Phe), hydrolyzed the tRNA selectively at the 3'-side of C63.
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PMID:Selective hydrolysis of tRNA by ethylenediamine bound to a DNA oligomer. 788 43

Ricin A-chin and alpha-sarcin are ribotoxins that inactivate eukaryotic ribosomes by modifying 28 S rRNA; ricin A-chain is an RNA N-glycosidase that depurinates the adenosine at position 4324 and alpha-sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of the adjacent guanosine (at position 4325). In cartoons of the secondary structure these two residues are seen to be embedded in a 17 base single-stranded loop over a seven base-pair helix. However, NMR spectroscopy of an oligoribonucleotide, a 29-mer that mimics the sarcin/ricin domain, indicates that the RNA has a compact conformation in which the guanosine at the position analogous to 4319 in 28 S rRNA is bulged out of what otherwise is an extended A-form helix. Since similar structural irregularities are used by proteins to bind to RNA, we have tested the effect of mutations of the bulged guanosine on recognition and covalent modification of the RNA by ricin A-chain and by alpha-sacrin. For the test a synthetic oligoribonucletide, a 35-mer, was used; the mutations were the deletion, the transition to adenosine, and the transversion to cytidine and uridine of the guanosine that is the analog of G4319. Each of the four mutations abolished cleavage og the RNA by alpha-sacrin, where depurination by ricin A-chain was little affected. Thus G4319 is an identity element for alpha-sacrin recognition. Analysis of the effect of alpha-sacrin on variant oligoribonucleotides in which additional bases were inserted between the identity element guanosine and the site of catalysis suggest that on binding to the RNA the toxin uses the guanosine for orientation and then cleaves at a fixed distance and at a fixed position in space.
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PMID:Determination of the 28 S ribosomal RNA identity element (G4319) for alpha-sarcin and the relationship of recognition to the selection of the catalytic site. 860 35

Ribozymes are sequences of catalytic RNA that are being evaluated as possible antisense therapeutics. This paper describes how capillary electrophoresis (CE) could be used to measure the catalytic rate of a synthetic hammerhead ribozyme in cleaving its substrate. This substrate was a synthetic full-RNA 17-mer, whereas the ribozyme was made up of a mixture of 37 2'-OH and 2'-OCH3 RNA nucleotides. After experimental conditions to exclude ribonuclease contamination were successfully met, different CE modes were tried out to separate the ribozyme from its substrate. Only the combination of chemical and thermal denaturation was adequate to disrupt strong secondary structures and to inhibit comigration of the two molecules. Cleavage kinetics were measured by continuous injection from the reaction vial into a polymer-filled capillary, and by determination of the area of the shrinking substrate peak. Compared to the well-established slab gel electrophoresis, CE is at least one order of magnitude faster, may be completely automated, allows easier and more precise quantitation of results, and, due to the small scale and self-contained nature of the apparatus, reduces health risks from dangerous chemicals. Unfortunately, UV detection in a 100-microm internal diameter capillary lacked the sensitivity to perform assays in the nanomolar range, which was necessary for a full Michaelis-Menten analysis.
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PMID:Capillary electrophoresis of RNA oligonucleotides: catalytic activity of a hammerhead ribozyme. 988 17

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. Residues Tyr47, His49, Glu95, Phe96, Pro97, Arg120, and His136 have been predicted to form the active site of restrictocin. In this study, we have individually mutated these amino acids to alanine to probe their role in restrictocin structure and function. The role of Tyr47, His49, Arg120, and His136 was further investigated by making additional mutants. Mutating Arg120 or His136 to alanine or the other amino acids rendered the toxin completely inactive, whereas mutating Glu95 to alanine only partially inactivated the toxin. Mutation of Phe96 and Pro97 to Ala had no effect on the activity of restrictocin. The Tyr47 to alanine mutant was inactive in inhibiting protein synthesis, and had a nonspecific ribonuclease activity on 28S rRNA similar to that shown previously for the His49 to Ala mutant. Unlike the His136 to Ala mutant, the double mutants containing Tyr47 or His49 mutated to alanine along with His136 did not compete with restrictocin to cause a significant reduction in the extent of cleavage of 28S rRNA. In a model of restrictocin and a 29-mer RNA substrate complex, residues Tyr47, His49, Glu95, Arg120, and His136 were found to be near the cleavage site on RNA. It is proposed that in restrictocin Glu95 and His136 are directly involved in catalysis, Arg120 is involved in the stabilization of the enzyme-substrate complex, Tyr47 provides structural stability to the active site, and His49 determines the substrate specificity.
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PMID:Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin. 1147 78

A double-stranded ribonuclease (Bm-dsRNase) was separated from the digestive juice of the silkworm larvae, Bombyx mori. The full-length cDNA was produced and sequenced using a 20 mer primer designed from the N-terminal sequence of the Bm-dsRNase. The cDNA had an ORF encoding 51 kDa precursor protein which can be divided into three domains: a signal peptide, an N-terminal propeptide and a mature Bm-dsRNase. The precursor has an Arg-Ser cleavage site, which produces the 43 kDa mature protein by post-translational processing. The 43 kDa protein had conserved catalytic amino acid residues which are also found in the active site of the Serratia marcescens dsRNase. Expression of the precursor occurred in the middle and posterior midgut tissues, starting from Day 1 of the fifth instar larvae. The 43 kDa protein was produced in this tissue from Day 2, and coincidentally secreted into the lumen containing digestive juice. This was supported by the immunohistochemical observation that the mature proteins were localized in the apical side of midgut cells for extracellular secretion.
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PMID:Molecular characterization of a cDNA encoding extracellular dsRNase and its expression in the silkworm, Bombyx mori. 1724 46

A novel protein family, designated hereafter as RNase kappa (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares > 98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subcloned into a variety of prokaryotic expression vectors, but most efforts to express it caused a severe toxic effect. On the other hand, the expression of the human RNase by the use of the methylotrophic yeast Pichia pastoris system resulted in the production of a highly active recombinant enzyme. Using a 30-mer 5'-end-labeled RNA probe as substrate, the purified enzyme seems to preferentially cleave ApU and ApG phosphodiester bonds, while it hydrolyzes UpU bonds at a lower rate. Based on amino acid sequence alignment and substrate specificity data, as well as the complete resistance of the recombinant protein to the placental ribonuclease inhibitor, we concluded that the human RNase kappa is a novel endoribonuclease distinct from other known ribonucleases.
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PMID:Molecular cloning and characterization of the human RNase kappa, an ortholog of Cc RNase. 1788 63

Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to form a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k2/K1/2 = 1.1 x 10(9) M-1 s-1). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a single-stranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex (E.S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.
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PMID:Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin. 1792 42

Recently we found that an antisense 2'-O-methyloligonucleotide, with two terpyridine*Cu(II) complexes at contiguous internal sites, was highly active as a site-specific (sequence-specific) artificial ribonuclease, with the activity derived from the cooperative action of the complexes. Two kinds of terpyridine-linked nucleosides were used for the construction of the RNA cleaver, including a uridine derivative with terpyridine attached to the 2'-oxygen via a short linker arm. In order to explore more efficient cleavers (practical cleavers), we have constructed a structurally similar cleaver (18-mer), but containing a novel 2'-carbon-branched uridine with a terpyridine group instead of the aforementioned 2'-oxygen-modified uridine. The reaction of a 10-fold excess of the target RNA 24-mer with the new agent, in the presence of Cu(II) ions, and at pH 7.5 and 37 degrees C, revealed that the substrate was cleaved in 92% yield after 5 h. Under similar conditions, the previous cleaver was less active and the cleavage yield was 61%.
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PMID:Development of site-specific artificial ribonucleases. 1802 57


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