Abstract
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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Selected References
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- Andreadis A., Hsu Y. P., Kohlhaw G. B., Schimmel P. Nucleotide sequence of yeast LEU2 shows 5'-noncoding region has sequences cognate to leucine. Cell. 1982 Dec;31(2 Pt 1):319–325. doi: 10.1016/0092-8674(82)90125-8. [DOI] [PubMed] [Google Scholar]
- Baldi M. I., Mattoccia E., Tocchini-Valentini G. P. Role of RNA structure in splicing: excision of the intervening sequence in yeast tRNA3leu is dependent on the formation of a D stem. Cell. 1983 Nov;35(1):109–115. doi: 10.1016/0092-8674(83)90213-1. [DOI] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
- Brown A., Pan C. J., Marzluff W. F. Methylation of ribonucleic acid in a cell-free system from mouse myeloma cells. Biochemistry. 1982 Aug 31;21(18):4303–4310. doi: 10.1021/bi00261a020. [DOI] [PubMed] [Google Scholar]
- Cech T. R. RNA splicing: three themes with variations. Cell. 1983 Oct;34(3):713–716. doi: 10.1016/0092-8674(83)90527-5. [DOI] [PubMed] [Google Scholar]
- Colby D. S., Schedl P., Guthrie C. A functional requirement for modification of the wobble nucleotide in tha anticodon of a T4 suppressor tRNA. Cell. 1976 Nov;9(3):449–463. doi: 10.1016/0092-8674(76)90090-8. [DOI] [PubMed] [Google Scholar]
- Engelke D. R., Gegenheimer P., Abelson J. Nucleolytic processing of a tRNAArg-tRNAAsp dimeric precursor by a homologous component from Saccharomyces cerevisiae. J Biol Chem. 1985 Jan 25;260(2):1271–1279. [PubMed] [Google Scholar]
- Fischhoff D. A., Waterston R. H., Olson M. V. The yeast cloning vector YEp13 contains a tRNALeu3 gene that can mutate to an amber suppressor. Gene. 1984 Mar;27(3):239–251. doi: 10.1016/0378-1119(84)90069-6. [DOI] [PubMed] [Google Scholar]
- Gamulin V., Mao J., Appel B., Sumner-Smith M., Yamao F., Söll D. Six Schizosaccharomyces pombe tRNA genes including a gene for a tRNALys with an intervening sequence which cannot base-pair with the anticodon. Nucleic Acids Res. 1983 Dec 20;11(24):8537–8546. doi: 10.1093/nar/11.24.8537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gefter M. L., Russell R. L. Role modifications in tyrosine transfer RNA: a modified base affecting ribosome binding. J Mol Biol. 1969 Jan 14;39(1):145–157. doi: 10.1016/0022-2836(69)90339-8. [DOI] [PubMed] [Google Scholar]
- Green C. J., Kammen H. O., Penhoet E. E. Purification and properties of a mammalian tRNA pseudouridine synthase. J Biol Chem. 1982 Mar 25;257(6):3045–3052. [PubMed] [Google Scholar]
- Greer C. L., Javor B., Abelson J. RNA ligase in bacteria: formation of a 2',5' linkage by an E. coli extract. Cell. 1983 Jul;33(3):899–906. doi: 10.1016/0092-8674(83)90032-6. [DOI] [PubMed] [Google Scholar]
- Greer C. L., Peebles C. L., Gegenheimer P., Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell. 1983 Feb;32(2):537–546. doi: 10.1016/0092-8674(83)90473-7. [DOI] [PubMed] [Google Scholar]
- Guthrie C., Seidman J. G., Altman S., Barrell B. G., Smith J. D., McClain W. H. Identification of tRNA precursor molecules made by phage T4. Nat New Biol. 1973 Nov 7;246(149):6–11. doi: 10.1038/newbio246006a0. [DOI] [PubMed] [Google Scholar]
- Hirsh D. Tryptophan transfer RNA as the UGA suppressor. J Mol Biol. 1971 Jun 14;58(2):439–458. doi: 10.1016/0022-2836(71)90362-7. [DOI] [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janner F., Vögeli G., Fluri R. The antisuppressor strain sin1 of Schizosaccharomyces pombe lacks the modification isopentenyladenosine in transfer RNA. J Mol Biol. 1980 May 15;139(2):207–219. doi: 10.1016/0022-2836(80)90305-8. [DOI] [PubMed] [Google Scholar]
- Johnson J. D., Ogden R., Johnson P., Abelson J., Dembeck P., Itakura K. Transcription and processing of a yeast tRNA gene containing a modified intervening sequence. Proc Natl Acad Sci U S A. 1980 May;77(5):2564–2568. doi: 10.1073/pnas.77.5.2564. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson P. F., Abelson J. The yeast tRNATyr gene intron is essential for correct modification of its tRNA product. Nature. 1983 Apr 21;302(5910):681–687. doi: 10.1038/302681a0. [DOI] [PubMed] [Google Scholar]
- Keith J. M., Winters E. M., Moss B. Purification and characterization of a HeLa cell transfer RNA(cytosine-5-)-methyltransferase. J Biol Chem. 1980 May 25;255(10):4636–4644. [PubMed] [Google Scholar]
- Koski R. A., Allison D. S., Worthington M., Hall B. D. An in vitro RNA polymerase III system from S. cerevisiae: effects of deletions and point mutations upon SUP4 gene transcription. Nucleic Acids Res. 1982 Dec 20;10(24):8127–8143. doi: 10.1093/nar/10.24.8127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koski R. A., Clarkson S. G., Kurjan J., Hall B. D., Smith M. Mutations of the yeast SUP4 tRNATyr locus: transcription of the mutant genes in vitro. Cell. 1980 Nov;22(2 Pt 2):415–425. doi: 10.1016/0092-8674(80)90352-9. [DOI] [PubMed] [Google Scholar]
- Kurjan J., Hall B. D., Gillam S., Smith M. Mutations at the yeast SUP4 tRNATyr locus: DNA sequence changes in mutants lacking suppressor activity. Cell. 1980 Jul;20(3):701–709. doi: 10.1016/0092-8674(80)90316-5. [DOI] [PubMed] [Google Scholar]
- Laten H., Gorman J., Bock R. M. Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. Nucleic Acids Res. 1978 Nov;5(11):4329–4342. doi: 10.1093/nar/5.11.4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee M. C., Knapp G. Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates. J Biol Chem. 1985 Mar 10;260(5):3108–3115. [PubMed] [Google Scholar]
- Liebman S. W., Srodulski Z., Reed C. R., Stewart J. W., Sherman F., Brennan G. Yeast amber suppressors corresponding to tRNA3Leu genes. J Mol Biol. 1984 Sep 15;178(2):209–226. doi: 10.1016/0022-2836(84)90140-2. [DOI] [PubMed] [Google Scholar]
- Mattoccia E., Baldi M. I., Pande G., Ogden R., Tocchini-Valentini G. P. Mutation in the a block of the yeast tRNAleu3 gene that allows transcription but abolishes splicing and 5'-end maturation. Cell. 1983 Jan;32(1):67–76. doi: 10.1016/0092-8674(83)90497-x. [DOI] [PubMed] [Google Scholar]
- McClain W. H., Seidman J. G. Genetic perturbations that reveal tertiary conformation of tRNA precursor molecules. Nature. 1975 Sep 11;257(5522):106–110. doi: 10.1038/257106a0. [DOI] [PubMed] [Google Scholar]
- Melton D. A., De Robertis E. M., Cortese R. Order and intracellular location of the events involved in the maturation of a spliced tRNA. Nature. 1980 Mar 13;284(5752):143–148. doi: 10.1038/284143a0. [DOI] [PubMed] [Google Scholar]
- Miller J. H., Albertini A. M. Effects of surrounding sequence on the suppression of nonsense codons. J Mol Biol. 1983 Feb 15;164(1):59–71. doi: 10.1016/0022-2836(83)90087-6. [DOI] [PubMed] [Google Scholar]
- Mitra S. K., Lustig F., Akesson B., Lagerkvist U. Codon-acticodon recognition in the valine codon family. J Biol Chem. 1977 Jan 25;252(2):471–478. [PubMed] [Google Scholar]
- Newman A. J., Ogden R. C., Abelson J. tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter. Cell. 1983 Nov;35(1):117–125. doi: 10.1016/0092-8674(83)90214-3. [DOI] [PubMed] [Google Scholar]
- Ng R., Domdey H., Larson G., Rossi J. J., Abelson J. A test for intron function in the yeast actin gene. Nature. 1985 Mar 14;314(6007):183–184. doi: 10.1038/314183a0. [DOI] [PubMed] [Google Scholar]
- Nishikura K., De Robertis E. M. RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. J Mol Biol. 1981 Jan 15;145(2):405–420. doi: 10.1016/0022-2836(81)90212-6. [DOI] [PubMed] [Google Scholar]
- Nishikura K., Kurjan J., Hall B. D., De Robertis E. M. Genetic analysis of the processing of a spliced tRNA. EMBO J. 1982;1(2):263–268. doi: 10.1002/j.1460-2075.1982.tb01157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ogden R. C., Lee M. C., Knapp G. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates. Nucleic Acids Res. 1984 Dec 21;12(24):9367–9382. doi: 10.1093/nar/12.24.9367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pearson D., Willis I., Hottinger H., Bell J., Kumar A., Leupold U., Söll D. Mutations preventing expression of sup3 tRNASer nonsense suppressors of Schizosaccharomyces pombe. Mol Cell Biol. 1985 Apr;5(4):808–815. doi: 10.1128/mcb.5.4.808. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peebles C. L., Gegenheimer P., Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
- Phizicky E. M., Schwartz R. C., Abelson J. Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. J Biol Chem. 1986 Feb 25;261(6):2978–2986. [PubMed] [Google Scholar]
- Raymond G. J., Johnson J. D. The role of non-coding DNA sequences in transcription and processing of a yeast tRNA. Nucleic Acids Res. 1983 Sep 10;11(17):5969–5988. doi: 10.1093/nar/11.17.5969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
- Sakano H., Shimura Y., Ozeki H. Selective modification of nucleosides of tRNA precursors accumulated in a temperature sensitive mutant of Escherichia coli. FEBS Lett. 1974 Nov 1;48(1):117–121. doi: 10.1016/0014-5793(74)81076-8. [DOI] [PubMed] [Google Scholar]
- Saneyoshi M., Oashi Z., Harada F., Nishimura S. Isolation and characterization of 2-methyladenosine from Escherichia coli tRNA Glu 2 , tRNA Asp 1 , tRNA His 1 and tRNA Arg . Biochim Biophys Acta. 1972 Feb 23;262(1):1–10. [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sharp P. A. On the origin of RNA splicing and introns. Cell. 1985 Sep;42(2):397–400. doi: 10.1016/0092-8674(85)90092-3. [DOI] [PubMed] [Google Scholar]
- Shaw K. J., Olson M. V. Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene. Mol Cell Biol. 1984 Apr;4(4):657–665. doi: 10.1128/mcb.4.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sprinzl M., Moll J., Meissner F., Hartmann T. Compilation of tRNA sequences. Nucleic Acids Res. 1985;13 (Suppl):r1–49. doi: 10.1093/nar/13.suppl.r1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sumner-Smith M., Hottinger H., Willis I., Koch T. L., Arentzen R., Söll D. The sup8 tRNALeu gene of Schizosaccharomyces pombe has an unusual intervening sequence and reduced pairing in the anticodon stem. Mol Gen Genet. 1984;197(3):447–452. doi: 10.1007/BF00329941. [DOI] [PubMed] [Google Scholar]
- Swerdlow H., Guthrie C. Structure of intron-containing tRNA precursors. Analysis of solution conformation using chemical and enzymatic probes. J Biol Chem. 1984 Apr 25;259(8):5197–5207. [PubMed] [Google Scholar]
- Weissenbach J., Dirheimer G. Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA3Arg. Biochim Biophys Acta. 1978 May 23;518(3):530–534. doi: 10.1016/0005-2787(78)90171-5. [DOI] [PubMed] [Google Scholar]
- Willis I., Hottinger H., Pearson D., Chisholm V., Leupold U., Söll D. Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor. EMBO J. 1984 Jul;3(7):1573–1580. doi: 10.1002/j.1460-2075.1984.tb02013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]