Figure 7
VapC-mt11 requires a GG sequence within the proper context. In vitro synthesized M. tuberculosis tRNAPro14 mutants were incubated with increasing amounts of recombinant VapC-mt11 (ratios of toxin:tRNA were 0:1, 5:1, 10:1, 15:1) for 3 h at 37 °C. Sizes for full-length and cleavage products (left). Cleavage assays with in vitro synthesized wild-type tRNAPro14 (A), tRNAPro14 point mutation (G −> A) of each G residue of the consensus GG sequence (blue shaded circles) mutants (B,C), tRNAPro14 mutant with point mutation 3′ of cut site (D) and tRNAPro14 mutants with point mutations within the anticodon sequence (E,F). Secondary structure of M. tuberculosis tRNAPro14 wild-type or mutants shown above gels. Anticodon sequence (shaded grey), base pairing represented as black dots (●), mutated bases (red), consensus sequence (shaded blue circles), wild-type cleavage site (red arrow), alternate cleavage site (green arrow), weak cleavage at wild-type cleavage site (small yellow arrow). All uncropped images shown in Supplementary Information Fig. 7.
