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)

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.
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PMID:Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro. 353 90

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.
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PMID:Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo. 353 24

The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro. This gene encodes a pre-tRNA which contains a 32-base intervening sequence. Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron. These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S. cerevisiae tRNA endonuclease and ligase. The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome. Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing. All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction. Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect. The implications of these data for tRNA endonuclease-substrate interactions are discussed.
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PMID:Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA. 353 25

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.
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PMID:Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem. 354 4

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

Strains of Saccharomyces cerevisiae have been constructed that possess temperature-sensitive defects in tRNA precursor (pre-tRNA) splicing and which also lack the processing endonuclease that acts at the 3'-terminus of 5 S rRNA and 35 S rRNA precursors (pre-rRNAs). The unspliced pre-tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre-tRNA splicing-defective strains with a functional pre-5 S RNA processing enzyme. The pre-RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse-labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild-type strains, an RNA 145 +/- 2 nucleotides in length.
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PMID:The effects of protein synthesis inhibition, and of mutations rna1.1 and rna82.1, on the synthesis of small RNAs in yeast. 355 24

In eukaryotes pre-tRNA species are processed at the 5' end by an endonuclease. Here we describe the first characterization of the structure of a eukaryotic pre-tRNA 5' processing endonuclease. The 5' pre-tRNAase, isolated from X. laevis ovaries, copurifies with a 16S macromolecular complex consisting of at least 14 polypeptides ranging in MW from about 20,000 to 32,000. These polypeptides comprise a cylindrical particle, apparently organized as a stack of four rings, similar or identical to a ubiquitous eukaryotic subcellular particle described in the literature over the past 15 years. Similar copurification is observed for the enzyme from HeLa cells, suggesting that the X. laevis enzyme is representative of a general class of eukaryotic pre-tRNA 5' processing nuclease.
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PMID:Eukaryotic pre-tRNA 5' processing nuclease: copurification with a complex cylindrical particle. 363 21

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

A DNA fraction enriched in tRNA genes has been prepared by CsCl density gradient centrifugation of Xenopus laevis DNA in the presence of actinomycin D. This DNA fraction was cut with the restriction endonuclease EcoRI and the fragments 800-900 base pairs in size were cloned into the plasmid pBR325. Recombinant DNAs were screened by hybridization to labeled tRNA and for the ability to support transcription in vitro. The entire sequence of one fragment was determined by sequencing the ends of an overlapping set of deletion fragments. A sequence homologous to tRNAVal from mammalian sources was found in this fragment and it was shown that this sequence corresponds to the region of the fragment that is transcribed. The cloned fragment was also transcribed in vivo after injection into X. laevis oocytes. The RNA that was synthesized in the oocytes was digested with ribonuclease T1 and the oligonucleotides were separated to produce a two-dimensional fingerprint. The results of the analysis of the oligonucleotides are consistent with the sequence determined for the tRNAVal gene. The X. laevis genome has 200-250 copies of the 892 base pair EcoRI fragment and additional copies of a 4100 base pair EcoRI fragment that each contain a tRNAVal gene. Digestion of X. laevis DNA with several other restriction endonucleases reveals that the cloned fragment that contains the tRNAVal gene is part of a longer sequence element that is tandemly repeated in the genome.
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PMID:Sequence and transcription of tRNAVal gene from Xenopus laevis. 380 87

We have previously reported that the primary transcript of the human tRNAMeti gene undergoes accurate processing to a mature 72-nucleotide species by activities present in the high speed supernatant of Xenopus laevis ovarian extracts (Zasloff, M., Santos, T., Romeo, P., and Rosenberg, M. (1982a) J. Biol. Chem. 257, 7857-7863). We now report the purification and characterization of the enzyme which processes the 3' terminus of the human pre-tRNAMeti species. The activity has been purified about 500-fold from a high speed supernatant of X. laevis ovarian extracts by standard methods. It appears to function as a single polypeptide with a molecular weight of about 97,400. The enzyme generates the mature 3' terminus with a single endonucleolytic cut, also yielding the intact 3' trailer. The endonuclease has a striking preference for the 5' processed pre-tRNAMeti, exhibiting little or no activity in vitro on the intact primary transcript. The enzyme acts similarly with the pre-tRNAAla species of Bombyx mori, suggesting that it possesses a broad substrate range. The requirement of the 3' processing endonuclease for a processed 5' terminus suggests that eukaryotic pre-tRNA processing should follow an ordered cutting sequence in vivo with processing of the 5' leader preceding 3' end maturation.
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PMID:Purification and characterization of an endonuclease from Xenopus laevis ovaries which accurately processes the 3' terminus of human pre-tRNA-Met(i) (3' pre-tRNase). 384 32


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