FIG 7.

Efficient cleavage of tRNA^fMet by VapC1^NTHi and VapC2^NTHi requires key base pairs in the anticodon stem structure. (A) Diagram showing anticodon stem-loop sequences and structures of tRNAs and mutants. Boxed base pairs indicate mutations made from either tRNA^Met-CAT or tRNA^fMet. (B) Differential hybridization Northern blot analysis of total RNAs isolated from E. coli BL21(DE3) cells expressing the VapC1 (left) or VapC2 (right) toxin (induced with 0.01% l-arabinose) and tRNAs (induced with 0.5 mM IPTG) as indicated, performed as described in Materials and Methods. The blots were hybridized with an oligonucleotide specific to both E. coli tRNA^fMet or tRNA^Met-CAT and the mutant tRNA, but with one nucleotide mismatch for the wild-type tRNA. After hybridization of the probe, blots were washed at 42°C and then again at 68°C. The image is a composite, as each tRNA required hybridization of a different oligonucleotide. Complete images may be seen in Fig. S1 in the supplemental material. (C) Quantification of cleaved tRNA/total tRNA from Northern blots, measured using ImageQuant. The data are averages for two biological replicates, and error bars represent standard deviations. *, P < 0.05; **, P < 0.01; N.S, not significant.