To develop a system for conditional amino acid misincorporation, we engineered tRNAs in the yeast Saccharomyces cerevisiae to be substrates of the rapid tRNA decay (RTD) pathway, such that they accumulate when RTD is turned off. We used this system to test the effects on growth of a library of tRNASer variants with all possible anticodons, and show that many are lethal when RTD is inhibited and the tRNA accumulates. Using mass spectrometry, we measured serine misincorporation in yeast containing each of six tRNA variants, and for five of them identified hundreds of peptides with serine substitutions at the targeted amino acid sites. Unexpectedly, we found that there is not a simple correlation between toxicity and the level of serine misincorporation; in particular, high levels of serine misincorporation can occur at cysteine residues without obvious growth defects. We also showed that toxic tRNAs can be used as a tool to identify sequence variants that reduce tRNA function. Finally, we generalized this method to another tRNA species, and generated conditionally toxic tRNATyr variants in a similar manner. This method should facilitate the study of tRNA biology and provide a tool to probe the effects of amino acid misincorporation on cellular physiology.

During tRNA maturation in yeast, aberrant pre-tRNAs are targeted for 3 ′ ′ ′ ′ ′ -5 ′ ′ ′ ′ ′ degradation by the nuclear surveillance pathway, and aberrant mature tRNAs are targeted for 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ degradation by the rapid tRNA decay (RTD) pathway. RTD is catalyzed by the 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonucleases Xrn1 and Rat1, which act on tRNAs with an exposed 5 ′ ′ ′ ′ ′ end due to the lack of certain body modifications or the presence of destabilizing mutations in the acceptor stem, T-stem, or tRNA fold. RTD is inhibited by mutation of MET22, likely due to accumulation of the Met22 substrate adenosine 3 ′ ′ ′ ′ ′ ,5 ′ ′ ′ ′ ′ bis-phosphate, which inhibits 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonucleases. Here we provide evidence for a new tRNA quality control pathway in which intron-containing pre-tRNAs with destabilizing mutations in the anticodon stem are targeted for Met22-dependent pre-tRNA decay (MPD). Multiple SUP4 οc anticodon stem variants that are subject to MPD each perturb the bulge-helix-bulge structure formed by the anticodon stem-loop and intron, which is important for splicing, resulting in substantial accumulation of end-matured unspliced pre-tRNA as well as pre-tRNA decay. Mutations that restore exon-intron structure commensurately reduce pre-tRNA accumulation and MPD. The MPD pathway can contribute substantially to decay of anticodon stem variants, since pre-tRNA decay is largely suppressed by removal of the intron or by restoration of exon-intron structure, each also resulting in increased tRNA levels. The MPD pathway is general as it extends to variants of tRNA Tyr(GUA) and tRNA Ser(CGA) . These results demonstrate that the integrity of the anticodon stem-loop and the efficiency of tRNA splicing are monitored by a quality control pathway.