Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

Mark Winey, Michael D. Mendenhall, Claudia M. Cummins, Michael R. Culbertson, Gayle Knapp

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


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.

Original languageEnglish
Pages (from-to)49-63
Number of pages15
JournalJournal of Molecular Biology
Issue number1
StatePublished - Nov 5 1986

Bibliographical note

Funding Information:
This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; National Institutes of Health grants GM26217 (M.R.C.) and GM30356 (G.K.). M.W. and C.M.C. were supported by Public Health Service training grants GM07215 and GM07133 in the Graduate Training Program in Molecular Biology and the Department of Genetics, respectively. Oligonucleotide synthesis performed by the University of Wisconsin Biotechnology Center was supported by National Institutes of Health grants SEG-SlO-RR01684 and NCI-CA-07175 We thank Jim Dahlberg and Peter Leeds for useful discussions and Mike Slater for valuable suggestions. We also thank Incell Corporation for synthesis of oligonucleotides. This is Laboratory of Genetics paper no. 2853.

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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