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 complete nucleotide sequences of human placenta, human liver, and bovine liver tRNAAsn have been determined. A comparison of these tRNA structures with the previously reported nucleotide sequences of rat liver and Walker 256 carcinosarcoma tRNAAns reveals that the primary nucleotide sequences of the major species of mammalian cytoplasmic tRNAasn are conserved in higher eucaryotes. The complete nucleotide sequence of these tRNAs is: pG-U-C-U-C-U-G-U-m1G-m2G-C-G-C-A-A-D-C-G-G-D-X-A-G-C-G-C-m2(2)G-psi-psi-C-G-G-C-U-Q(G)-U-U-t6A-A-C-C-G-A-A-A-G-m7G-D-U-G-G-U-G-G-Z-psi-C-G-m1A-G-C-C-C-A-C-C-C-A-G-G-G-A-C-G-C-C-AOH where X is 3-(3-amino-3-carboxyl-n-propyl)uridine, Q is 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, Z is an unknown modified nucleotide, and Q(G) represents the replacement of Q nucleoside by G nucleoside in Walker 256 carcinosarcoma tRNAAsn. These primary structures were determined by combined use of the 3H- and 32P-post-labeling techniques. Sequences were compared by tritium nucleoside trialcohol analysis, completed RNAase T1 digestion followed by 3H-labeled fingerprinting on polyethylenimine-impregnated cellulose by two-dimensional thin-layer chromatography (TLC), and polyacrylamide gel electrophoresis of either 5'-32P- and/or 3'-[32P]pCp-labeled tRNA after partial ribonuclease digestions.
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PMID:Structural comparison of human, bovine, rat, and Walker 256 carcinosarcoma asparaginyl-tRNA. 678 75

N-Acetylvalyl-tRNA1Val (AcVal-tRNA1Val) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of 300-400 nm. To identify the crosslinked nucleotide in 16S RNA. AcVal-tRNA1Val-16S [32P]RNA was digested completely with RNase T1 and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2. The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNA1Val, C1400. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNA1Val-16S RNA prepared from unlabeled ribosomes. This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked T1 oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleotide of tRNA1Val, C-A-C-C-U-C-C-C-U-cmo5U-A-C-m6A-A-G39(cmo5U, 5-carboxymethoxyuridine). A similar study of the crosslinked oligonucleotide revealed that the residue covalently bound to 16S was cmo5U34, the 5' or wobble base of the anticodon. The adduct is believed to result from formation of a cyclobutane dimer between cmo5U34 of tRNA1Val and C1400 of the 16S RNA.
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PMID:Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues. 681 60

An enzyme complex is a multifunctional catalytic unit that efficiently associates substrates with functionally related enzymes. The enzyme complex provides for the cellular regulation of enzymatic activities by physical interaction of the proteins with each other and by prior alteration of one enzyme's substrate by a related enzyme. Such regulatory abilities may go awry in neoplasia. Components of the protein biosynthetic machinery, such as aminoacyl-tRNA synthetases, have been thought to exist freely in the cytoplasm. However, high-molecular-weight enzyme complexes with aminoacyl-tRNA synthetase activities have been found in mammalian cells. We have been the first to report that the mammalian cell enzymes responsible for modification of tRNA occur in enzyme complexes (molecular weight 900000 daltons) associated with aminoacyl-tRNA synthetases and that the activities of these enzymes differ in normal and leukemic cells. Thus the enzymes responsible for the methylation of tRNA occur in enzyme complexes that provide efficient maturation of tRNA and possible regulation of protein synthesis. In FLC cells a unique enzyme complex composed of tRNA-methyltransferase and aminoacyl-tRNA synthetase activities has also been shown to contain a specific ribonuclease activity and a cysteine-tRNA sulfurtransferase activity. Sulfurtransferase activity has been characterized and optimized for its tRNA and cysteine substrates and mercaptoethanol and cation cofactors. Abnormal activity of this enzyme during neoplasia could result in improper acylation of tRNA and/or infidelity of coding by tRNA. Specific RNase is important in the sizing of percursor tRNA into mature tRNA. Results showed that this sizing was dependent upon the presence of the enzyme complex and the length of the incubation time. Many of the 20 aminoacyl-tRNA synthetases are also found in the complex. Electron microscopy has verified the subunit nature of the complex, seen previously by density gradient centrifugation and gel filtration. Three subunits, each of 300 000 daltons, comprise a complex approximately 200 A in diameter.
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PMID:Processing of tRNA is accomplished by a high-molecular-weight enzyme complex. 684 94

The hydrolysis of several tRNAs by an endonuclease extracted from the venom of Naja oxiana and specific for double-stranded, or at least highly ordered, regions has been studied under various experimental conditions. It is shown that the hydrolysis patterns of yeast tRNAPhe, tRNAVal and tRNAAsp in the isolated state are similar, most of the cuts occurring in the anticodon and acceptor stems. Ionic conditions are able to modify the hydrolysis pattern. The origin of these modifications is discussed. The protection against ribonuclease action, afforded to tRNAPhe, tRNAVal and tRNAAsp by the cognate aminoacyl-tRNA synthetase, is analyzed. It is shown that in all cases the anticodon stem is protected. The 3'-terminal region does not seem to be tightly engaged in the complex with the aminoacyl-tRNA synthetase. These results are discussed in the light of information on contact areas previously obtained by ultraviolet cross-linking techniques. The effects of the small ligands (ATP and amino acid) on the protection afforded to the tRNA by the cognate synthetase, have been studied. In the valine and aspartic acid systems, ATP induced a modification of the tRNA-enzyme complex leading to differences in the hydrolysis pattern of the 3'-accepting region. The effects of aminoacylation on the cleavage of tRNAPhe, tRNAVal and tRNAAsp were also studied. Whereas no modification of the cleavage map was observed in the aspartic system, aminoacylation resulted in slight but significant modifications of the hydrolysis pattern for tRNAPhe and tRNaVal in the 3'-terminal region.
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PMID:Comparison of the hydrolysis patterns of several tRNAs by cobra venom ribonuclease in different steps of the aminoacylation reaction. 691 54

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteine, the most highly purified 75Se-fractions were greater than 100-fold purified relative to 35S. These fractions contained less than 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.
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PMID:Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver. 692 51

Transfer RNA molecules have been labeled with 32P at the 5' or 3' end and digested with cobra venom ribonuclease, which preferentially cuts double-stranded regions. The products of yeast tRNAPhe and tRNAVal were analyzed by high-resolution gel electrohporesis. In the free state, these tRNAs were cut predominantly in the acceptor and anticodon stems. Minor cuts occurred in the T psi stem in tRNAVal. The topography of zones interacting with their cognate synthetases was studied by determining the tRNA regions shielded by protein. Nearly 100% protection was found in the anticodon and acceptor stem of tRNAVal, while in tRNAPhe only the stem of the anticodon was protected. Noncognate interactions between tRNAPhe and tryptophanyl-tRNA synthetase from beef pancreas were examined. The beef enzyme did not protect tRNAPhe despite the fact that efficient misaminoacylation occurred. The pattern of shielding obtained for each tRNA-synthetase complex was compared with the results of direct ultraviolet cross-linking experiments with these complexes.
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PMID:Partial digestion of tRNA--aminoacyl-tRNA synthetase complexes with cobra venom ribonuclease. 701 69

The presence of ribonuclease inhibitor and/or the activity of class I ribonucleases can conveniently be measured, at all stages of purification, by a highly sensitive assay based on the loss of radioactivity during the concomitant hydrolysis of tRNA and small amounts of 14C-labeled aminoacyl-tRNA. The rapid, economical assay, which is readily adaptable to homologous tRNA substrates, eliminates the necessity of filtration, centrifugation and ultraviolet spectroscopy measurements required by most other assays and is particularly suitable for multiple samples and kinetic measurements.
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PMID:A procedure for the rapid preparation of 14C-aminoacyl-transfer RNA and its use in the assay of class I ribonucleases and ribonuclease inhibitor. 701 24

In order to undertake structural and functional studies on the 3'-terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl-tRNA synthetase, we have developed large-scale methods for purifying the tRNA-like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA-like fragment. Starting from 1.5 g turnip yellow mosaic virus, one obtains 400 mg RNA, which is partially digested by T1 ribonuclease and which yields 1-2 mg pure tRNA-like fragment after three chromatographic steps: two filtrations on Ultrogel ACA 54 and one reverse-phase chromatography (RPC 5) in the presence of urea. A method has been worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous upon polyacrylamide gel electrophoresis under denaturing conditions. The isolated tRNA-like structure can be valylated to an extent of 100% in the presence of purified yeast valyl-tRNA synthetase with kinetic parameters resembling those of the tRNAVal aminoacylation.
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PMID:Large-scale purification of the 3'-OH-terminal tRNA-like sequence (n = 159) of turnip-yellow-mosaic-virus RNA. 706

Cytoplasmic 10S ribonucleoprotein (iRNP) is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). This ribonucleoprotein has now been resolved into protein and RNA components by DEAE-cellulose chromatography in the absence of both K+ and Mg2+ ions. These cations are required for maintaining the nucleoprotein structure of iRNP. Incubation of the dissociated protein and RNA components in the presence of K+ and Mg2+ at 35 degrees C reconstitutes a 10 S particle which is indistinguishable from native iRNP with respect to the elution profile by gel filtration, UV spectra, buoyant density, resistance to pancreatic RNase, and ability to inhibit exogenous mRNA translation in vitro. Chick muscle tRNA and globin mRNA could not form an RNP complex with the protein moieties of iRNP. The separated proteins, unlike iRNA and iRNP, do not inhibit mRNA translation. Their function may be to protect iRNA from ribonuclease digestion, since iRNP is ribonuclease-resistant. The ability to dissociate the iRNP particle and to specifically reconstitute it from the separated components indicates that it is a unique cellular entity which is distinct from other ribonucleoproteins.
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PMID:The translational inhibitor 10 S cytoplasmic ribonucleoprotein of chick embryonic muscle. Dissociation and reassociation. 728 68

The isolated ribonuclease (RNase) H domain of human immunodeficiency virus type 1 (HIV-1) is enzymatically inactive. The incorporation of the putative substrate binding site of Escherichia coli RNase HI (amino acid residues 76-102, the alpha c-helix and adjacent loop region) into the equivalent position of the RNase H domain of HIV-1 resulted in a highly active hybrid protein dependent on Mn2+. Similar restoration of RNase H activity has been observed when histidine residues are added to either the N- or C-terminus of the HIV-1 RNase H domain. The hybrid HIV-1/E. coli RNase H protein is approximately 10-fold more active than HIV-1 reverse transcriptase and 30-fold more active than the histidine-tagged proteins, indicating that the alpha c-helix and adjacent loop region of E. coli RNase HI is an excellent substrate binding region because of its sequence and/or location. The RNase H hybrid produced the same specific cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse transcriptase, showing that substrate binding and specificity are separable and that the specificity determinants are at least partially, if not totally, contained in the amino acid sequence of the hybrid protein derived from HIV-1 reverse transcriptase.
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PMID:Construction of an enzymatically active ribonuclease H domain of human immunodeficiency virus type 1 reverse transcriptase. 753 Mar 60


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