Abstract
Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.
Highlights
Most classes of cellular RNA from all organisms contain post-transcriptionally modified nucleotides
The Test Systems—Mature transfer tRNA contains a large variety of modified nucleotides
Occurring tRNAs contain a variety of modified nucleotides
Summary
Most classes of cellular RNA (tRNA, mRNA, rRNA, and small nuclear RNA) from all organisms contain post-transcriptionally modified nucleotides. In truA mutant cells, which are unable to modify the uridines at positions 38, 39, and 40, a decrease in the rate of the aminoacyl-tRNA selection step during translation depending on the identity of the tRNA has been demonstrated [13]. This observation could explain the pleiotropic phenotype of truA mutant cells, like derepression of his, leu, and ilv operons, reduction of growth, and polypeptide chain elongation rates (reviewed in Ref. 3). It has not been established whether these phenotypes are linked to the lack of ⌿ in anticodon arm of the tRNA or to the absence of the TruA protein per se
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