Summary tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Anticodon breakage inhibits protein synthesis, which in turn results in growth arrest or cell death. The eukaryal ribotoxin PaT elaborated by Pichia acaciae inhibits growth of Saccharomyces cerevisiae via cleavage of tRNAGln(UUG). We find that recombinant PaT incises a synthetic tRNAGln(UUG) stem-loop RNA by transesterification at a single site 3′ of the wobble uridine, yielding 2′,3′-cyclic phosphate and 5′-OH ends. Incision is suppressed by replacing the wobble nucleobase with adenine or guanine. We report the crystal structure of PaT, which unveils a novel fold and active site, essential constituents of which are illuminated by mutagenesis. Pichia acaciae evades self toxicity via a distinctive intracellular immunity protein, ImmPaT, which binds PaT and effaces nuclease activity. Our results highlight the evolutionary diversity of tRNA restriction and immunity systems.

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