Summary
We have cloned and sequenced the wild-type and suppressor alleles of the S. pombe sup8 tRNA gene. The wild-type allele has a leucine UAA anticodon and the suppressor (sup8-e) carries the opal suppressor anticodon UCA. The gene has a 16 base pair intervening sequence that, in the RNA, is predicted to form a secondary structure which involves base pairing to the 5′, rather than the usual 3′ side of the 5′ splice site. When incubated in Saccharomyces cerevisiae cell-free extracts both alleles are efficiently transcribed, the 5′ leader and 3′ trailer sequences are removed and CCA is added to the 3′ processed end; however, the intervening sequence is not excised. This finding implies that the structural requirements of the splicing endonucleases in the two yeasts have diverged. No other tRNA genes with related sequences were detected in S. pombe DNA by hybridization, suggesting that other UUA isoacceptors may be structurally dissimilar to sup8 or that the UUA codon may be decoded by a UUG leucine isoacceptor.
Similar content being viewed by others
References
Clewell DB (1972) Nature of ColEl plasmid replication in the presence of chloramphenicol. J Bacteriol 110:667–676
Egel R, Kohli J, Thuriaux P, Wolf K (1980) Genetics of the fission yeast Schizosaccharomyces pombe. Annu Rev Genet 14:77–108
Gamulin V, Mao J, Appel B, Sumner-Smith M, Yamao F, Söll D (1983) Six Schizosaccharomyces pombe tRNA genes including a gene for a tRNALys with an intervening sequence which cannot base-pair with the anticodon. Nucl Acids Res 11:8537–8546
Gutz H, Heslof H, Leupold U, Loprieno N (1974) Schizosaccharomyces pombe. In: King RC (ed) Handbook of genetics, vol 1. Plenum Press, New York, pp 394–446
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Hawthorne DC, Leupold U (1974) Suppressors in yeast. Curr Top Microbiol Immunol 64:1–47
Hofer F, Hollenstein H, Janner F, Minet M, Thuriaux P, Leupold U (1979) The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe. I. sup3 and sup9. Curr Genet 1:45–61
Hottinger H, Pearson D, Yamao F, Gamulin V, Cooley L, Cooper T, Söll D (1982) Nonsense suppression in Schizosaccharomyces pombe: The S. pombe sup3-e tRNA SerUGA gene is active in S. cerevisiae. Mol Gen Genet 188:219–224
Johnson PF, Abelson J (1983) The yeast tRNATyr gene intron is essential for correct modification of its tRNA product. Nature 302:681–687
Mao J, Schmidt O, Söll D (1980) Dimeric transfer RNA precursors in S. pombe. Cell 21:509–516
Messing J, Vieira J (1982) A new pair of M13 vectors selecting either DNA strand of double-digest restriction fragments. Gene 19:269–278
Messing J, Gronenborn B, Müller-Hill B, Hofschneider PH (1977) Filamentous coliphage M13 as a cloning vehicle: Insertion of a HindIII fragment of the lac regulatory region in M13 replicative form in vivo. Proc Natl Acad Sci USA 74:3642–3646
Munz P, Amstutz H, Kohli J, Leupold U (1982) Recombination between dispersed serine tRNA genes in Schizosaccharomyces pombe. Nature 300:225–231
Munz P, Dorsch-Häsler K, Leupold U (1983) The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe. II. sup8 and sup10. Curr Genet 7:101–108
Munz P, Leupold U, Agris P, Kohli J (1981) In vivo decoding rules in Schizosaccharomyces pombe are at variance with in vitro data. Nature 294:187–188
Nishikura K, Kurjan J, Hall BD, DeRobertis E (1982) Genetic analysis of the processing of a spliced tRNA. EMBO J 1:263–268
Pearson D, Willis I, Hottinger H, Bell J, Kumar J, Leupold U, Söll D (1984) Mutations preventing expression of sup3 tRNASer nonsense suppressors of S. pombe EMBO J (in press)
Peebles C, Gegenheimer P, Abelson J (1983) Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell 32:535–536
Raymond GJ, Johnson JD (1983) The role of non-coding DNA sequences in transcription and processing of a yeast tRNA. Nucl Acids Res 11:5969–5988
Roberts TM, Swanberg SL, Poteete A, Riedel G, Backman K (1980) A plasmid cloning vehicle allowing a positive selection for inserted fragments. Gene 12:123–127
Wetzel R, Kohli J, Altruda F, Söll D (1979) Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe. Mol Gen Genet 172:221–228
Willis I, Hottinger H, Pearson D, Chisholm V, Leupold U, Söll D (1984) Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor. EMBO J 3:1573–1580
Winter G, Fersht AR, Wilkinson AJ, Zoller M, Smith M (1982) Redesigning enzyme structure by site-directed mutagenesis: Tyrosyl tRNA synthetase and ATP binding. Nature 299:756–758
Author information
Authors and Affiliations
Additional information
Communicated by E. Bautz
Rights and permissions
About this article
Cite this article
Sumner-Smith, M., Hottinger, H., Willis, I. et al. The sup8 tRNALeu gene of Schizosaccharomyces pombe has an unusual intervening sequence and reduced pairing in the anticodon stem. Mol Gen Genet 197, 447–452 (1984). https://doi.org/10.1007/BF00329941
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00329941