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The nucleotide sequences of a Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9‐e) and of 12 in vivo‐generated mutant genes, which have lost the ability to suppress UGA mutations, have been determined. Analysis of the expression of these genes in Saccharomyces cerevisiae in vitro and in vivo systems has revealed defects in tRNA gene transcription and precursor tRNA processing. Single base changes in the D‐loop, the intron and the extra arm affect the efficiency of splicing of the tRNA precursors while an anti‐codon stem mutation may affect the accuracy of this process. Two mutations which occur in the intervening sequence of the sup9‐e gene allow an alternate tRNA base pairing configuration. Transcription of the sup9‐e gene and of the adjacent tRNAMet gene (located 7 bp downstream) is essentially abolished in vivo by a G–‐A19 mutation in the tRNASer gene, suggesting that tRNAMet may be derived solely via processing of the tRNASer‐tRNAMet dimeric precursor.

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Suppression of nonsense codons in Schizosaccharomyces pombe by sup3-e tRNAUGA or sup3-i tRNAUAA is reduced or abolished by mutations within the suppressor locus. Twenty-five suppressor-inactive sup3-e genes and thirteen mutant sup3-i genes were isolated from S. pombe genomic clone banks by colony hybridization. Sequence analysis of these revertant alleles corroborates genetic evidence for mutational hotspots within the sup3 tRNA gene. Fifteen types of point mutations or insertions were found. Many of these replace bases which are highly or completely conserved in eucaryotic tRNA genes. Transcription of the altered sup3 genes in a Saccharomyces cerevisiae extract enabled the identification of mutations which affect the rate of 5'-end maturation or splicing of the tRNA precursors or both. A total of seven mutations were found which alter transcriptional efficiencies. Of these, five are located outside the internal transcription control regions.The genetic analysis of tRNA-mediated nonsense suppression is well advanced in procaryotes and in the lower eucaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe (11,44,45). Fine-structure maps have been constructed for a number of suppressor loci in these organisms by examining the recombination frequencies between mutations within a given tRNA gene (14,20,27,37). We are pursuing the biochemical characterization of defined mutations at the sup3-e and sup9-e loci of S. pombe (20).These loci encode tRNAUGA genes, both of which have recently been cloned and sequenced (21, 49). Although located on different chromosomes, these genes share an almost identical dimeric tRNA gene arrangement; the serine tRNA gene is separated by seven base pairs from an initiator tRNAMet gene. These dimeric genes are transcribed into approximately 190-nucleotide-long precursors that differ by only a single base at the tip of the extra arm of the serine tRNA and several bases outside the tRNA coding regions. Mature tRNAs are produced via a complex processing pathway which includes the excision of an intervening sequence from the serine tRNA half of the precursor.In this paper, we report the characterization of 38 in vivo-derived sup3 revertant alleles by cloning and sequence analysis. They contain 15 types of point mutations or singlebase insertions within the sup3 tRNAser gene. Cloned alleles were transcribed in an Saccharomyces cerevisiae extract, allowing the identification of mutations affecting tRNA gene transcription and 5'-end maturation and splicing of the resulting transcripts. MATERIALS AND METHODSIsolation and sequence analysis of sup3 alleles. The Schizosaccharomyces pombe strains used in this work have been described by Hofer et al. (20) Gergen et al. (17). The hybridization probe consisted of the 1.0-kilobase HindIII-BamHI fragment containing the sup3-e gene (21) and was labeled to a specific activity of 106 to 108 cpm/,ug with T4 DNA polymerase and [a-32P]dATP (3,000 Ci/mmol). Plasmid DNA was prepared from strongly hybridizing colonies and digested with HindIII and EcoRI. ...

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