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. 1990 Aug;10(8):4379–4383. doi: 10.1128/mcb.10.8.4379

Construction of an opal suppressor by oligonucleotide-directed mutagenesis of a Saccharomyces cerevisiae tRNA(Trp) gene.

A L Atkin 1, K L Roy 1, J B Bell 1
PMCID: PMC360993  PMID: 2370870

Abstract

In vitro mutagenesis was used to create putative opal suppressor alleles of a tRNA(Trp) gene of Saccharomyces cerevisiae. The construct with the requisite anticodon change did not result in an active suppressor, whereas when a second change was introduced into the portion of the gene encoding the intron, an active and specific opal suppressor was produced. We propose that the secondary structure of transcripts from the first mutant may prevent efficient pre-tRNA processing, whereas normal processing occurs with the double mutant.

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Selected References

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  1. Dingermann T., Nerke K., Blöcker H., Frank R. Structural requirements for the synthesis of tRNATrp from Dictyostelium discoideum in yeast. Biochimie. 1988 Jun;70(6):711–719. doi: 10.1016/0300-9084(88)90099-5. [DOI] [PubMed] [Google Scholar]
  2. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Greer C. L., Söll D., Willis I. Substrate recognition and identification of splice sites by the tRNA-splicing endonuclease and ligase from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):76–84. doi: 10.1128/mcb.7.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kim D., Johnson J. Construction, expression, and function of a new yeast amber suppressor, tRNATrpA. J Biol Chem. 1988 May 25;263(15):7316–7321. [PubMed] [Google Scholar]
  6. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
  7. Mathison L., Winey M., Soref C., Culbertson M. R., Knapp G. Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Oct;9(10):4220–4228. doi: 10.1128/mcb.9.10.4220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Pure G. A., Robinson G. W., Naumovski L., Friedberg E. C. Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene. J Mol Biol. 1985 May 5;183(1):31–42. doi: 10.1016/0022-2836(85)90278-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Reyes V. M., Abelson J. Substrate recognition and splice site determination in yeast tRNA splicing. Cell. 1988 Nov 18;55(4):719–730. doi: 10.1016/0092-8674(88)90230-9. [DOI] [PubMed] [Google Scholar]
  16. Stewart T. S., Söll D., Sharp S. Point mutations in the 5' ICR and anticodon region of a Drosophila tRNAArg gene decrease in vitro transcription. Nucleic Acids Res. 1985 Jan 25;13(2):435–447. doi: 10.1093/nar/13.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Strobel M. C., Abelson J. Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo. Mol Cell Biol. 1986 Jul;6(7):2663–2673. doi: 10.1128/mcb.6.7.2663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Strobel M. C., Abelson J. Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA. Mol Cell Biol. 1986 Jul;6(7):2674–2683. doi: 10.1128/mcb.6.7.2674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stucka R., Feldmann H. Structure of a Saccharomyces cerevisiae gene encoding minor (AGY)tRNA(Ser). Nucleic Acids Res. 1988 Apr 25;16(8):3583–3583. doi: 10.1093/nar/16.8.3583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Szekely E., Belford H. G., Greer C. L. Intron sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae. J Biol Chem. 1988 Sep 25;263(27):13839–13847. [PubMed] [Google Scholar]
  21. Wallace R. B., Miyada C. G. Oligonucleotide probes for the screening of recombinant DNA libraries. Methods Enzymol. 1987;152:432–442. doi: 10.1016/0076-6879(87)52050-x. [DOI] [PubMed] [Google Scholar]
  22. Weiss W. A., Friedberg E. C. Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene. J Mol Biol. 1986 Dec 20;192(4):725–735. doi: 10.1016/0022-2836(86)90024-0. [DOI] [PubMed] [Google Scholar]
  23. van Tol H., Beier H. All human tRNATyr genes contain introns as a prerequisite for pseudouridine biosynthesis in the anticodon. Nucleic Acids Res. 1988 Mar 25;16(5):1951–1966. doi: 10.1093/nar/16.5.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]

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