The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNA Le 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 tRNASuPS3 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 tRNASuPS3. 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.Intervening sequences (IVS), or introns, have been found in the genes which encode the major classes of eucaryotic RNA, both nuclear and organellar. Recently, the mechanisms by which introns are removed from the primary RNA transcript have been the focus of intense investigation (reviewed in references 7, 14, and 51). However, relatively few experiments have elucidated a biological function for an IVS separate from the role of intron sequence or structure in the splicing process.Saccharomyces cerevisiae affords an ideal system for investigating intron function. A number of yeast nuclear mRNA and tRNA genes containing introns have been cloned. These genes can be mutated in vitro and the mutant phenotype assayed by introducing the mutated genes into yeast cells by chromosomal integration. Using these methods, Ng et al. (40) showed that precise removal of the S. cerevisiae actin gene intron did not affect the production of actin mRNA or actin protein in vivo. Cells containing only the intron-deleted actin gene exhibit a normal growth rate and normal meiotic products upon sporulation.In contrast, Johnson and Abelson (22) found that precise removal of the intron from an S. cerevisiae suppressor tRNA gene has profound effects on the phenotype of the mature suppressor tRNA product. Comparing strains containing the tyrosine-inserting ocher suppressor tRNASUP6 and its introndeleted counterpart SUP6A32, they found that SUP6A32 was inefficient in suppressing ocher nonsense mutations.When in vivo-labeled tRNATYr from the two strains was examined, SUP6-specific RNA from the deletion strain its total structure or some specific feature of the IVS itself, is recognized a...