FIGURE 2.
The γ-toxin is an endonuclease that cleaves tRNAGlumcm5s2UUC, tRNALysmcm5s2UUU, and tRNAGlnmcm5s2UUG. (A) SDS-PAGE analysis of γ-toxin-GST fraction (lane 1), further purified 53-kD γ-toxin- GST (lane 2), and GST (lane 3). Proteins were visualized by silver staining. (B,C) Northern blot analysis of wild-type S. cerevisiae tRNA (5 μg) incubated with 1 μg of γ-toxin-GST fraction (lane 1), 53-kDa γ-toxin-GST (lane 2), or GST (lane 3) for 10 min at 30°C. The filter was probed by using an oligonucleotide complementary to the 3′- (B) or the 5′-part (C) of tRNAGlumcm5s2UUC. (D) Reactions containing the indicated concentration of γ-toxin-GST fraction or GST, and 5 μg of wild-type, elp3Δ, or trm9Δ total tRNA was incubated for 10 min at 30°C. Samples were analyzed by Northern blots; the identity of each signal is indicated on the left. (E) A wild-type S. cerevisiae strain CY4029 (W303-1A SSD1-v1) carrying the indicated high copy (h.c.) plasmid was serially diluted, spotted onto a YEPD plate or a YEPD plate supplemented with crude zymocin, and incubated for 3 d at 25°C. The elp3Δ and trm9Δ strains carrying the empty h.c. vector were included on the plates.