Figure 4. DNA Damaging Agents Result in tRNA Cleavage and Operon Activation.
(A) After growing wild-type and ΔrecA FLAG3-rsr strains in MMC for the indicated times, FLAG3-Rsr, LexA, RecA, and RpoA (loading control) were detected on immunoblots. Asterisk, RecA fragment. (B–D) After growing wild-type and ΔrecA strains in MMC, northern analysis was performed to detect 5′ halves of the indicated tRNAs.
(E–H) RNA from strains grown in MMC was subjected to northern analysis to detect 5′ halves of the indicated tRNAs (lanes 3–18). Lanes 1 and 2, RNA from wild-type cells carrying empty vector or pRtcRΔN, respectively. In (E), the bottom panel is a lighter view of the 5′ halves. In (H), red lines denote fragments differing in mobility in ΔtruA and ΔtruA Δpnp strains.
(I) After treating FLAG3-rsr strains with bleomycin or MMS for 0.5, 1, 2, or 3 h, lysates were subjected to immunoblotting to detect FLAG3-Rsr and RplE.
(J) After growth of the indicated strains in 0 (top) and 1 μg/mL MMC (bottom panel) for 2 h, serial 10-fold dilutions were spotted on Luria-Bertani broth (LB) agar and grown at 37°C.
(K) Aliquots of the cells in (J) were plated on LB agar and colonies counted to determine the fraction of surviving cells. Data are mean (n = 3) ± SEM. p values were calculated with two-tailed unpaired t tests. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.
See also Figure S3.