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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The divalent metal ion binding site and binding constant of ribonuclease HI from Escherichia coli were investigated by observing chemical shift changes using 1H-15N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by 15N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide 15N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg2+, Ca2+, or Ba2+. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (KD = 2-4 mM for the current divalent ions).
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PMID:Binding of metal ions to E. coli RNase HI observed by 1H-15N heteronuclear 2D NMR. 166 22

The two sequences that define the self-cleaving elements from the genomic and antigenomic RNA of hepatitis delta virus were folded into secondary structures with similar features. Evidence in support of the two models was obtained from limited ribonuclease digestion of genomic and antigenomic RNA fragments containing the sequence 3' of the cleavage site. Under conditions where the rates of self-cleavage are enhanced by addition of 5 M urea (2-10 mM Mg2+ at 37 degrees C), ribonucleases T1, U2, A and V1 generated digestion patterns consistent with the proposed RNA structures. The evidence for a relatively stable structure in urea when Mg2+ is present suggests that denaturant-enhanced rates of self-cleavage could result from destabilization of competing inactive structures.
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PMID:Evidence that genomic and antigenomic RNA self-cleaving elements from hepatitis delta virus have similar secondary structures. 192 26

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.
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PMID:Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation. 233 46

We identified a Mg2+ dependent 5' exo-ribonuclease and an RNA ligase in cell-free extracts of Trypanosome brucei. The exo-ribonuclease in S100 or nuclear extracts, removes about 20 nts from the 5' end of SP6 derived capped as well as uncapped RNA and then stops. In contrast to the activity of the exo-ribonuclease on capped SP6 mini-exon transcripts, the exonuclease cannot degrade trypanosome-derived mini-exon transcripts or the mini-exon located at hsp 70 mRNAs. We therefore assume that the four secondary base modifications adjacent to the mini-exon cap, generated in vivo, confer resistance to the exo-ribonuclease. After exonuclease shortening of SP6 transcripts, an RNA ligase catalizes intramolecular ligation, generating a 3'-5' phosphodiester bond in a Mg2+ and ATP dependent reaction.
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PMID:A 5' exo-ribonuclease and RNA ligase of T. brucei. 246 Aug 26

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

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

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

Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.
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PMID:Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins. 310 66

A technique for isolating defined fragments of a large RNA has been developed and applied to a ribosomal RNA. A section of the Escherichia coli rrnB cistron corresponding to the S8/S15 protein binding domain of 16S ribosomal RNA was cloned into a single-stranded DNA phage; after hybridization of the phage DNA with 16S RNA and digestion with T1 ribonuclease, the protected RNA was separated from the DNA under denaturing conditions to yield a 345-base RNA fragment with unique ends (bases 525-869 in the 16S sequence). The secondary structure of this fragment was determined by mapping the cleavage sites of enzymes specific for single-stranded or double-helical RNA. The fragment structure is almost identical with that proposed for the corresponding region of intact 16S RNA on the basis of phylogenetic comparisons [Woese, C. R., Gutell, R., Gupta, R., & Noller, H. (1983) Microbiol. Rev. 47, 621-669]. We conclude that this section of RNA constitutes an independently folding domain that may be studied in isolation from the rest of the 16S RNA. The structure mapping experiments have indicated several interesting features in the RNA structure. (i) The largest bulge loop in the molecule (20 bases) contains specific tertiary structure. (ii) A region of long-range secondary structure, pairing bases about 200 residues apart in the sequence, can hydrogen bond in two different mutually exclusive schemes. Both appear to exist simultaneously in the RNA fragment under our conditions. (iii) The long-range secondary structure and one adjacent helix melt between 37 and 60 degrees C in the absence of Mg2+, while the rest of the structure is quite stable.
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PMID:Secondary structure of a 345-base RNA fragment covering the S8/S15 protein binding domain of Escherichia coli 16S ribosomal RNA. 390 98


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