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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.
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PMID:Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain. 133 59

We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.
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PMID:Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates. 1989 Oct 53

To investigate the mechanism by which the purified Xenopus tRNA splicing endonuclease recognizes its splice sites, we utilized yeast pre-tRNA(3Leu) and pre-tRNA(Phe) variants constructed by in vitro mutagenesis. We found that the endonuclease interacts with conserved features of the mature tRNA domain. In particular, U8 and C56 may be examples of contact points between protein and RNA. Given that there are no conserved sequences at the splice junctions, the specificity of cutting at both splice sites is determined by the length of the anticodon stem. Although in general, the sequence of the intron is unimportant for splicing, there are some structural requirements.
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PMID:Site selection by the tRNA splicing endonuclease of Xenopus laevis. 318 Feb 24

We have examined the substrate requirements for efficient and accurate splicing of tRNA precursors in Saccharomyces cerevisiae. The effects of Schizosaccharomyces pombe tRNASer gene mutations on the two steps in splicing, intron excision and joining of tRNA halves, were determined independently by using partially purified splicing endonuclease and tRNA ligase from S. cerevisiae. Two mutations (G14 and A46) reduced the efficiency of excision and joining in parallel, whereas two others (U47:7 and C33) produced differential effects on these two steps; U47:7 affected primarily the excision reaction, and C33 had a greater impact on ligation. These data indicate that endonuclease and ligase recognize both common and unique features of their substrates. Another two mutations (Ai26 and A37:13) induced miscutting, although with converse effects on the two splice sites. Thus, the two cutting events appear to be independent. Finally, we suggest that splice sites may be determined largely through their position relative to sites within the tRNA-like domain of the precursors. Several of these important sites were identified, and others are proposed based on the data described here.
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PMID:Substrate recognition and identification of splice sites by the tRNA-splicing endonuclease and ligase from Saccharomyces cerevisiae. 355 Apr 27

An intron-containing tobacco tRNA(Tyr) precursor synthesized in a HeLa cell nuclear extract has been used to develop a cell-free processing and splicing system from wheat germ. Removal of 5' and 3' flanking sequences, accurate excision of the intervening sequence, ligation of the resulting tRNA halves, addition of the 3'-terminal CCA sequence and modification of seven nucleosides were achieved in appropriate wheat germ S23 and S100 extracts. The maturation of pre-tRNA(Tyr) in these extracts resembles the pathway observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast in that processing of the flanks precedes intron excision. Most of the modified nucleosides (m2(2) G, psi 35, psi 55, m7G and m1A) are introduced into the intron-containing pre-tRNA with mature ends, whereas two others (m1G and psi 39) are only found in the mature tRNA(Tyr). Processing and splicing proceed very efficiently in the wheat germ extracts, leading to complete maturation of 5' and 3' ends followed by about 65% conversion to mature tRNA(Tyr) under our standard conditions. The activity of the wheat germ endonuclease is stimulated 3-fold by the non-ionic detergent Triton X-100. All previous attempts to demonstrate the presence of a splicing endonuclease in wheat germ had failed (Gegenheimer et al., 1983). Hence, this is the first cell-free plant extract which supports pre-tRNA processing and splicing in vitro.
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PMID:A cell-free plant extract for accurate pre-tRNA processing, splicing and modification. 367 5

The splicing of tRNA precursors is essential for the production of mature tRNA in organisms from all major phyla. In yeast, the tRNA splicing endonuclease is responsible for identification and cleavage of the splice sites in pre-tRNA. We have cloned the genes encoding all four protein subunits of endonuclease. Each gene is essential. Two subunits, Sen2p and Sen34p, contain a homologous domain of approximately 130 amino acids. This domain is found in the gene encoding the archaeal tRNA splicing endonuclease of H. volcanii and in other Archaea. Our results demonstrate that the eucaryal tRNA splicing endonuclease contains two functionally independent active sites for cleavage of the 5' and 3' splice sites, encoded by SEN2 and SEN34, respectively. The presence of endonuclease in Eucarya and Archaea suggests an ancient origin for the tRNA splicing reaction.
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PMID:The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases. 920 Jun 3

The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron. The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases. We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron. One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon-intron boundaries, are used for recognition of either one or the other cleavage site. The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place. These results support a two-active-site model for the eucaryal endonuclease.
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PMID:The eucaryal tRNA splicing endonuclease recognizes a tripartite set of RNA elements. 920 Jun 4

At least two separate enzymes, an endonuclease and a ligase, appear to be involved in tRNA splicing in halophilic archaea. We have identified and partially characterized a splicing ligase activity in cell extracts of Haloferax volcanii that can ligate deproteinized exon products generated in a separate endonuclease reaction. As in vitro transcribed partial intron-deleted derivative of H. volcanii elongator tRNA(Met) is used as substrate for the endonuclease. The ligase can also join the two exons that are independently eluted from the gels. This ligase activity is observed at a range (50 mM to 2.8 M) of monovalent cations in the assays, but is abolished when the enzyme preparations are depleted of the monovalent cations. In contrast, H. volcanii splicing endonuclease has been reported to require divalent cations and is inhibited by monovalent cations. Our endonuclease assays confirm these reports, and also show that the endonuclease is not permanently inactivated even in high monovalent cation containing extracts. The ligase activity in the extracts does not appear to require any divalent cation or exogenously added source of energy or phosphate.
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PMID:RNA splicing ligase activity in the archaeon Haloferax volcanii. 929 9

We report the complete sequence of two cosmids, SPBC19C7 (34815 bp insert, Accession No. AL023859) and SPBC15D4 (33203 bp insert, Accession No. AL031349), localized on chromosome II of the S. pombe genome. Twelve open reading frames (ORFs) were identified in SPBC19C7 and 16 in SPBC5D4. Two known genes were found on each cosmid: cyr1 and uve1 on SPBC19C7, encoding adenylate cyclase and a UV-endonuclease, respectively, and gpt and pho2 on SPBC15D4, encoding an N-acetylglucosamine-1-phosphate transferase and a4-nitrophenylphosphatase, respectively. Five ORFs similar to known proteins were found on SPBC19C7, and six on SPBC15D4. They include putative genes for a ubiquitin protein ligase, a prolyl-tRNA synthetase, a tRNA splicing endonuclease, a voltage-gated chloride channel, a mannosyl transferase, a kinesin-like protein, a histone transcriptional regulator, an N-acetyltransferase, a cystathionine gamma-synthase and a TFIID subunit. Two ORF products of SPBC15D4 do not have clear homologues: one encodes a putative transcriptional regulator with a binuclear zinc domain and the other a protein with six transmembrane domains. Two ORFs from SPBC15D4 are similar to unknown ORFs, one from Saccharomyces cerevisiae and the other from Caenorhabditis elegans. Finally, two ORFs of SPBC19C7 and six of SPBC15D4 correspond to orphan genes. The frequent occurrence of introns and the short and degenerated intron-exon boundaries consensus sequences significantly complicated ORF predictions. Two potential ORF-free regions spanning several kb were predicted, and a clustering of ORFs transcribed in the same orientation was observed.
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PMID:Sequence analysis of two cosmids from the right arm of the Schizosaccharomyces pombe chromosome II. 1066 67

tRNA splicing endonuclease is required to produce mature tRNAs from intron-containing tRNA precursors. To characterize the structural features of plant endonuclease, we have isolated a cDNA and a corresponding genomic DNA clone from libraries of Arabidopsis thaliana which encode a putative subunit of the endonuclease. The gene product has an apparent mass of 27 kDa and contains a homologous domain of approximately 130 amino acids at the C-terminal region commonly found in other eucaryal and archaeal counterparts. Southern hybridization analysis of Arabidopsis genomic DNA utilizing the cDNA clone as probe indicates the presence of at least two related genes.
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PMID:Molecular cloning and characterization of a nuclear gene encoding a putative subunit of tRNA splicing endonuclease from Arabidopsis thaliana. 1078 Apr 79


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