Figure 3.
VapC-mt4 requires a consensus sequence within a proper structural context. Cleavage assays comparing wild-type VapC-mt4 cleavage of M. tuberculosis tRNASer24 (A) to a mutant that retains the consensus sequence but removes all base pairing in the stem (B). Mutated bases (blue), cleavage site (yellow arrow), consensus sequence (red), anticodon (gray shaded), base-pairing represented as black dots (•). In vitro synthesized mutant and wild-type tRNASer24 were incubated with increasing amounts of VapC-mt4 (ratios of toxin to RNA were 0:1, 1.25:1, 2.5:1 and 5:1). Control reactions on the right contained the highest concentration of VapC-mt4 preincubated with VapB antitoxin or EDTA before addition of the respective tRNAs. Reactions were incubated at 37°C for 3 h. Sizes of full length and cleaved tRNA products on the left. Convention when numbering tRNA bases dictates that the anticodon is numbered bases 34–36. However, for the actual base numbers for the anticodon are 35–37.