To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suJ8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G-U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suJ8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the + 1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppressor alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affect tRNA splicing.Splicing of transfer RNA in Saccharomyces cerevisiae requires three separate enzymes: a detergent-extractable endonuclease, a soluble ligase, and a 2'-phosphatase (26). The endonuclease cleaves the pre-tRNA, releasing the intron as a linear molecule (25). The ligase joins the half molecules through an ATP-dependent, three-step mechanism involving cyclic phosphodiesterase, polynucleotide kinase, and RNA ligase activities (12, 28). Finally, the residual 2'-phosphate at the splice site is removed to produce a mature, primary tRNA sequence.The splicing enzymes of S. cerevisiae process all introncontaining intermediates in the synthesis of 10 of the 46 cytoplasmic tRNA isoacceptors (14, 23). The enzymes form an in vitro complex composed minimally of the endonuclease and the ligase (11). The ligase, and by extension probably the complex as well, is localized in vivo in associatio...

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G. C base pairs, with all base pairs in each run having identical orientation. Long consecutive G. C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.