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:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During tRNA biosynthesis the 5'-leader sequences in precursor tRNAs are removed by the ribonucleoprotein RNase P, an enzyme whose RNA moiety is required for activity. To clarify some aspects of the enzyme mechanism, we examined substrate binding and product formation with mutant precursor tRNAs. Mutations G-1----A or U-2----C in the Schizosaccharomyces pombe sup3-e tRNASer, which cause mispairing at or near the top of the acceptor stem, prevent the removal of the 5'-leader sequences by Saccharomyces cerevisiae RNase P. Equilibrium binding studies involving specific gel retardation of RNase P-precursor tRNA complexes showed that complexes with wild-type and A-1 and C-2 mutant precursor tRNAs had very similar dissociation constants (average Kd for sup3 = 1.5 +/- 0.2 nM). Thus, the 5'-terminal nucleotides of mature tRNA, on the 3' proximal side of the RNase P cleavage site, affect the enzyme's catalytic function but not substrate binding. The catalytic integrity of the RNA component of RNase P is not essential for binding of tRNA precursors, as demonstrated by gel retardation of micrococcal nuclease-inactivated enzyme. This suggests a possible role for the protein component of the enzyme in substrate binding. Upon restoration of base pairing to the acceptor stem in the A-1 or C-2 mutants, we found that, in addition to a requirement for pairing at these positions, conservation of the wild-type first and second nucleotides of the tRNA was necessary to obtain maximal cleavage by RNase P. This indicates a distinct sequence preference of this enzyme.
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PMID:Yeast RNase P: catalytic activity and substrate binding are separate functions. 327 10

A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors. We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA. This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity. The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation. The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.
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PMID:RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components. 353 97

Sodium cyanate is a selective in vivo inhibitor of protein synthesis in a variety of mammalian tumor cells without a corresponding effect on the normal tissues of tumor-bearing animals. The in vivo decrease of protein synthesis observed 4 h post-NaOCN i.p. administration in the murine P388 leukemia cell cannot be explained by decreased amino acid pools in the mouse peritoneal cavity. In addition, the decrease in protein synthesis observed with NaOCN in isolated P388 cells was shown not to be secondary to (a) alterations in the kinetics of amino acid transport or (b) effects on total nucleotide pools. The incorporation of [14C]phenylalanine in P388 cell-free lysates from NaOCN-pretreated mice was significantly decreased to approximately 55% of control lysates in the presence of exogenous amino acids. The addition of exogenous calf liver tRNA to the lysates did not alter this result. However, no difference was observed in polyuridylic acid-directed [14C]phenylalanine incorporation into polypeptides in micrococcal nuclease-treated P388 lysates from NaOCN-pretreated or control mice. Quaternary initiation complex (48S) formation and mRNA synthesis were found to be significantly decreased by 35 and 38%, respectively, in P388 cells from NaOCN-pretreated mice. DNA synthesis was decreased by 66% of control at 1 h and 62% at 4 h post-NaOCN i.p. administration. No apparent effect with NaOCN was observed on total RNA synthesis in P388 cells. These results suggest that the decrease in P388 cell protein synthesis observed with NaOCN in vivo appears to be due to alterations manifested in the synthesis of cellular mRNA and protein synthesis initiation processes. NaOCN does not appear to affect the P388 cell ribosomal machinery, tRNA, or protein synthesis elongation processes.
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PMID:Mechanism of decrease of protein synthesis by sodium cyanate in murine P388 leukemia cells. 362 Nov 95

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.
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PMID:Experimental methyl mercury neurotoxicity: locus of mercurial inhibition of brain protein synthesis in vivo and in vitro. 384 56

The known methods of enzymatic phosphorylation with [(32)P]phosphate of the 3'- or 5'-hydroxyl group of an oligonucleotide have been applied to oligonucleotides derived from Mycoplasma tRNA(Phe). The fingerprints obtained by both methods are very similar to each other and to that of uniformly labelled tRNA. The sequence of some oligonucleotides was determined by partial digestion of the 3'-phosphorylated fragment with spleen phosphodiesterase and of the corresponding 5'-phosphorylated fragment with venom phosphodiesterase.
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PMID:Sequence studies of nonradioactive Mycoplasma tRNA Phe with the aid of polynucleotide phosphorylase and polynucleotide kinase. 444 33

A low molecular weight RNA species, in the 70-90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography. This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash. The iRNA at the concentration of 20-80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e. chick muscle poly(A)+mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature. The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA. The iRNA does not appear to be double-stranded RNA. It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.
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PMID:Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle. 617 43

Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases. Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models. These methods allow detailed kinetic analyses on small amounts of chromatin. Two biological systems were studied. 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities. 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied. DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin. Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results. These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases. Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed. These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.
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PMID:Nuclease sensitivity of chromatin containing active genes: kinetic analyses utilizing continuous elution of digestion products from an ultrafiltration cell. 627 9

We have studied the structure of tandemly repetitive alpha-satellite chromatin (alpha-chromatin) in African green monkey cells (CV-1 line), using restriction endonucleases and staphylococcal nuclease as probes. While more than 80% of the 172-base-pair (bp) alpha-DNA repeats have a HindIII site, less than 15% of the alpha-DNA repeats have an EcoRI site, and most of the latter alpha-repeats are highly clustered within the CV-1 genome. EcoRI and HindIII solubilize approximately 8% and 2% of the alpha-chromatin, respectively, under the conditions used. EcoRI is thus approximately 30 times more effective than HindIII in solubilizing alpha-chromatin, with relation to the respective cutting frequencies of HindIII and EcoRI on alpha-DNA. EcoRI and HindIII solubilize largely non-overlapping subsets of alpha-chromatin. The DNA size distributions of both EcoRI- and HindIII-solubilized alpha-chromatin particles peak at alpha-monomers. These DNA size distributions are established early in digestion and remain strikingly constant throughout the digestion with either EcoRI or HindIII. Approximately one in every four of both EcoRI- and HindIII-solubilized alpha-chromatin particles is an alpha-monomer. Two-dimensional (deoxyribonucleoprotein leads to DNA) electrophoretic analysis of the EcoRI-solubilized, sucrose gradient-fractionated alpha-oligonucleosomes shows that they do not contain "hidden" EcoRI cuts. Moreover, although the EcoRI-solubilized alpha-oligonucleosomes contain one EcoRI site in every 172-bp alpha-DNA repeat, they are completely resistant to redigestion with EcoRI. This striking difference between the EcoRI-accessible EcoRI sites flanking an EcoRI-solubilized alpha-oligonucleosome and completely EcoRI-resistant internal EcoRI sites in the same alpha-oligonucleosome indicates either that the flanking EcoRI sites occur within a modified chromatin structure or that an altered nucleosome arrangement in the vicinity of a flanking EcoRI site is responsible for its location in the nuclease-sensitive internucleosomal (linker) region. Analogous redigestions of the EcoRI-solubilized alpha-oligonucleosomes with either HindIII, MboII or HaeIII (both before and after selective removal of histone H1 by an exchange onto tRNA) produce a self-consistent pattern of restriction site accessibilities. Taken together, these data strongly suggest a preferred nucleosome arrangement within the EcoRI-solubilized subset of alpha-oligonucleosomes, with the centers of most of the nucleosomal cores being approximately 20 bp and approximately 50 bp away from the nearest EcoRI and HindIII sites, respectively, within the 172-bp alpha-DNA repeat. However, as noted above, the clearly preferred pattern of nucleosome arrangement within the EcoRI-solubilized alpha-oligonucleosomes is invariably violated at the ends of every such alpha-oligonucleosomal particle, suggesting at least a partially statistical origin of this apparently non-random nucleosome arrangement.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Nucleosome arrangement in green monkey alpha-satellite chromatin. Superimposition of non-random and apparently random patterns. 631 39

A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.
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PMID:Characterization of messenger RNA by direct translation from agarose gels. 666 May 15

A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation. Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes. Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes. Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes. It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures. The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2. Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes. This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract. The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins. Partial purification of the activating factor(s) has been achieved by ion exchange chromatography.
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PMID:Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract. 686 64


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