Intracellular γ-toxin expression in S. cerevisiae reduces cell viability and the level of tRNA^Glu_mcm⁵s²UUC. (A) Structure of mcm⁵s²U. An elp3Δ mutant lacks the entire mcm⁵ side-chain (box with solid lines) (Huang et al. 2005), whereas a trm9Δ strain lacks the indicated methyl group (box with dotted lines) (Kalhor and Clarke 2003). The formation of the 2-thio group appears not to be affected in the elp3 and trm9 mutants (Huang et al. 2005; data not shown). (B) Wild-type, elp3Δ, and trm9Δ strains with an integrated P_GAL1-γ-toxin construct were shifted from raffinose (uninduced) to galactose (induced) containing medium. The ratio of viable to total cells was determined at indicated time points. (C) Northern blot analysis of total RNA isolated from the induced or uninduced cultures in B. The tRNA^Glu_mcm⁵s²UUC signal was quantified and normalized to the tRNA^Ser_CGA signal. Below each lane is the normalized value expressed relative to the corresponding value at time point 0, which is set to 1.

The γ-toxin is an endonuclease that cleaves tRNA^Glu_mcm⁵s²UUC, tRNA^Lys_mcm⁵s²UUU, and tRNA^Gln_mcm⁵s²UUG. (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 tRNA^Glu_mcm⁵s²UUC. (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.

K. lactis γ-toxin cleaves tRNA^Glu_mcm⁵s²UUC, tRNA^Lys_mcm⁵s²UUU, and tRNA^Gln_mcm⁵s²UUG between position 34 and 35. (A) γ-toxin-GST– treated (+) or mock-treated (−) wild-type total tRNA was reverse transcribed by using ³²P-labeled oligonucleotides complementary to the 3′-end of tRNA^Glu_mcm⁵s²UUC, tRNA^Lys_mcm⁵s²UUU, or tRNA^Gln_mcm⁵s²UUG. The sequence ladder was derived from a gene coding for tRNA^Glu_mcm⁵s²UUC. Arrowheads indicate reverse transcripts induced by γ-toxin treatment. (B) The purified 5′-half of tRNA^Glu_mcm⁵s²UUC, tRNA^Lys_mcm⁵s²UUU, or tRNA^Gln_mcm⁵s²UUG was treated with the indicated enzyme(s) before ligation and RT-PCR amplification. (C) The sequence shared between tRNA^Glu_mcm⁵s²UUC, tRNA^Lys_mcm⁵s²UUU, and tRNA^Gln_mcm⁵s²UUG in the anticodon region, and the site of γ-toxin cleavage.