Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To make strong statements about possible tertiary structure or the relative stability of regions of secondary structure, the structure-probing experiments must go further than single-hit reactions. Some elements of the environment of the RNA molecule must be altered systematically. Knowledge of the effects of ions or other interacting factors on the activity or physical parameters (e.g., NMR and melting cooperativity) of the RNA help in experimental design. For example, the copious work on tRNA(Phe) compared the crystal and solution structures and allowed the direct correlation of Mg2+ stabilization of the tertiary structure of that molecule. Figure 3 demonstrates that pre-tRNA(Leu-3) responds to Mg2+ depletion in the same manner as detected by the appearance of highly sensitive RNase cleavage sites in the D and T psi C loops. Similar experiments titrating polyamine concentrations suggested that secondary structure was more efficiently stabilized by polyamines than by Mg2+. The variation of Mg2+ concentrations has been used to gain additional information about other RNA structures. Others have used protein-RNA interactions to approach the question of the functional structure of a RNA (for examples, see Ref. 3). Thus, the ideal parameters to choose would be those known to affect the function of the RNA. The variation of Mg2+ and polyamine concentrations would minimally suggest regions of greater or lesser secondary or tertiary structure stability.
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PMID:Enzymatic approaches to probing of RNA secondary and tertiary structure. 248 14

We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR). In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+. In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro. In contrast, high concentrations of Zn2+ or Cu2+ suppress protein binding and inhibit the processing reaction. The purified exoribonuclease polynucleotide phosphorylase (E.C.2.7.7.8) is not efficient in processing the pet D 3' IR-RNA precursor, whereas Escherichia coli ribonuclease II rapidly processes the pet D IR-RNA precursor to a product of a size similar to that of the mature 3' IR-RNA, but also rapidly degrades the mature RNA in the absence of chloroplast extract. We therefore conclude that the maturation of the pet D mRNA in vitro requires specific chloroplast enzymes which process the mRNA 3' end precursor in the absence of efficient transcription termination. The chloroplast enzyme activities are biochemically distinct from their bacterial counterparts. We also note that specific chloroplast components may be required to stabilize the mature pet D mRNA 3' end against further exonucleolytic degradation.
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PMID:Chloroplast mRNA 3' end maturation is biochemically distinct from prokaryotic mRNA processing. 248 89

Native small nuclear ribonucleoproteins (snRNPs) purified by several conventional procedures or reconstituted in vitro have no ribonuclease activity. However, when these same snRNPs are centrifuged in cesium chloride gradients at low [Mg2+] and in the presence of sarkosyl, an endoribonuclease is unmasked at the density of core particles (i.e. containing only the set of low molecular weight proteins common to all snRNPs), while an inhibitory component is released in soluble form. The nature of this inhibitor was not further investigated and the molecular events underlying this inhibition/activation process remained only a matter of speculation. On the other hand, evidence was obtained that the nuclease activity is carried by B-B' on the basis of its comigration with B-B' as well as with two of their cleavage products after SDS/polyacrylamide gel electrophoresis of snRNP proteins. One was identified by a B-B'-specific monoclonal antibody. Another one, especially prominent and migrating between D and E core proteins, was identified as the N-terminal half of B-B' by microsequence analysis. Although tightly associated with core snRNPs, the activity is not dependent upon the presence of an snRNA. For the time being, the functional significance of this nuclease remains entirely elusive.
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PMID:B-B' proteins from small nuclear ribonucleoproteins have an endoribonuclease catalytic domain inactive in native particles. 252 74

The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg2+. In this paper, the effect of Zn2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg2+. Phosphorylation patterns of viral and other proteins depend on the divalent cation present. In the presence of Zn2+, phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. Our results indicate the activation of more than one virus-associated protein kinase by Zn2+. The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn2+. The destabilization leads to a substantially increased permeability of virus particles to ethidium bromide and RNase, concomitant with decreased infectivity of the sample. This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. High-performance liquid chromatography-purified viral protein VP2 is phosphorylated by the released enzymes on serine, threonine, and tyrosine in the presence of Zn2+. We suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus.
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PMID:Poliovirus-associated protein kinase: destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+. 254 9

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63,000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity.
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PMID:Poly(A) polymerase from Vigna unguiculata seedlings. A bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. 255 12

We have developed and characterized cell-free systems active in translation from unfertilized eggs, 30-min zygotes and hatched blastulae of the sea urchin Strongylocentrotus purpuratus. The ion concentrations selected for preparation of the lysates were 150 mM-K+, 40 mM-Na+, 40 mM-Cl-, 5 x 10(-7) M free Ca2+ and 1 mM free Mg2+. It was necessary to include the ribonuclease inhibitor RNas in the preparations to obtain full activity consistently. The pH optimum was 7.2 and was extremely sharp for the three S. purpuratus lysates. The temperature optima of the three lysates were remarkably similar to those of the intact unfertilized egg and embryos. Lysates from unfertilized egg and 30-min zygotes showed a temperature optimum at 15 degrees C. The hatched blastula lysate showed a broader temperature optimum with a shift to about 20 degrees C. The optimized lysates incorporated radiolabelled amino acids into polypeptides for up to 90 min. The polypeptides synthesized ranged in Mr from 200,000 to 20,000, suggesting that the mRNA in the lysates was intact and capable of directing the synthesis of complete polypeptides. Furthermore, the three lysates were capable of initiation, as demonstrated by inhibition of initiation using the inhibitors edeine and 7-methylguanosine 5'-triphosphate (m7GTP). At 15 degrees C, the transit times for the three lysates were: unfertilized egg, 40 min; 30-min zygotes and hatched blastula lysates, 20 min. These transit times are similar to those of intact eggs and embryos, and significantly, reflect the two-fold increase in elongation rate seen following fertilization in intact embryos. Thus, these lysates display many features and characteristic responses typical of intact eggs and embryos, indicating that the lysates should be useful tools for the analysis of translation control in early embryogenesis.
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PMID:Characterization of translation systems in vitro from three developmental stages of Strongylocentrotus purpuratus. 270

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO2. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA(Glu), ATP, Mg2+, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[3H]glutamate or 1-[14C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[14C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the alpha subgroup of purple bacteria.
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PMID:Distribution of delta-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups. 278 25

Extracts of Rad+ and radiation-sensitive (rad) mutants of the yeast Saccharomyces cerevisiae were examined for total Mg2+-dependent alkaline deoxyribonuclease activity and the presence of a nuclease that crossreacts immunologically with an antiserum raised against an endo-exonuclease from Neurospora crassa, an enzyme exhibiting both deoxyribo- and ribonuclease activities. No significant differences were observed in total deoxyribonuclease activity between Rad+ and rad mutants. The antibody precipitable activity, however, was found to be 30%-40% of the total alkaline deoxyribonuclease activity in logarithmically growing Rad+ cells. Extracts of stationary phase cells were lacking in antibody precipitable activity. Using immunoblot methods, a 72 kDa crossreacting protein was identified from logarithmically growing cells that was absent from stationary phase cells. In all radiation-sensitive mutants examined, except rad52, at least 20% of total activity was precipitable. Extracts from logarithmically growing rad52 mutants, including a rad52::LEU2 insertion mutant, exhibited less than 10% of the Rad+ precipitable activity; however, some crossreacting material was detected. Although, the level of endo-exonuclease activity is influenced by the RAD52 gene, it is not the product of this gene. The total deoxyribonuclease and the antibody precipitable endo-exonuclease activities were also followed during meiosis. Unlike the Rad+ strain which had previously been shown to have increased levels of total and immunoprecipitable endo-exonuclease as cells underwent meiosis, the rad52 mutant exhibited no increases in either category of nuclease activity. Given the importance of the RAD52 gene in repair, recombination and mutagenesis, the endo-exonuclease may be a significant component of these processes.
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PMID:An endo-exonuclease activity of yeast that requires a functional RAD52 gene. 283 Apr 67

To facilitate further studies of flavivirus transcription, cell extraction methods and in vitro reaction conditions which increased West Nile virus (WNV) RNA-dependent RNA polymerase activity were determined. Subcellular fractions from WNV-infected BHK-21/W12 cells were characterized with regard to their protein and RNA content and in vitro polymerase activity. In both a cytoplasmic fraction, designated S1, and a fraction enriched for outer nuclear membranes, designated S2, seven virus-specific proteins, NS5 (96 kilodaltons [kDa]), NS3 (67 kDa), E (48 kDa), NS1 (47 kDa), ns4a (26 kDa), ns2a (17 kDa), and ns2b (14.5 kDa), were detected. The fractions also contained virus-specific RNA and cellular rRNA and mRNA. Polymerase activity in S1 and S2 fractions from WNV-infected cells was concentrated by pelleting and consisted of two types of enzyme activities: the WNV RNA-dependent RNA polymerase and terminal transferases of cellular origin. Enhanced levels of WNV polymerase activity were obtained from these cell fractions by altering several of the in vitro reaction conditions. Although Mg2+ was the divalent cation preferred by WNV polymerase, virus-specific in vitro transcription was detected at reduced levels when Mn2+ (0.05 or 0.5 mM) was present as the sole divalent cation. Product analysis revealed that the viral polymerase incorporated radiolabeled ribonucleotides into three distinct RNA species. Free single-stranded genome-sized RNA which was LiCl insoluble and RNase sensitive was found by fingerprint analysis to have an oligonucleotide pattern similar to that of WNV genomic RNA. RNA molecules which comigrated as a broad band near the top of the gel were separable into LiCl-insoluble, partially RNase-sensitive replicative-intermediate RNA and LiCl-soluble, RNase-resistant replicative-form RNA. The cellular transferases added UMP or AMP residues to the 3'-termini of cellular mRNA, tRNA, and 18S and 28S rRNA. Although a cellular terminal transferase has been reported to function in initiation of poliovirus transcription, no labeling of the WNV RNA by either of these cellular enzymes was detected. Therefore, they appear to play no specific role in flavivirus RNA synthesis.
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PMID:Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. 302 63

Calcium-activated neutral protease (CANP) has been purified from the human placenta by chromatographic procedures. The purified enzyme is a heterodimer with one subunit of mol. wt 70 000 and another of mol.wt 32 000. It is a thiol protease, active at pH 7.5 at 30 degrees C in the presence of calcium. Half-maximal activation of the enzyme occurred with 800 microM Ca2+.Zn2+ (2 mM), ethylenediaminetetraacetic acid (EDTA)(5 mM) and ethyleneglycol-bis-N,N,N',N'-tetraacetic acid (EGTA)(2 mM) inhibited the enzyme, while Mg2+ (0.5 mM to 5 mM) had no effect on the enzyme in the presence of calcium. Mn2+ and Ca2+ activated the enzyme synergistically. CANP coexists with its endogenous inhibitor in the human placenta. The inhibitor is a protein, inactivated by trypsin and unaffected by RNase, DNase, acid and heat treatments; it inhibits the enzyme probably by interacting with the enzyme molecule itself rather than by sequestering calcium ions.
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PMID:Calcium-activated neutral protease from human placenta: purification and characterization. 308 82


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