Figure 2. VapC-mt4 targets a specific subset of tRNAs in M. tuberculosis.

(a–c) In vitro VapC-mt4 cleavage assays showing the three tRNAs from among the 13 consensus-containing M. tuberculosis tRNAs tested that are cleaved to completion: tRNA^Ala2 (a), tRNA^Ser26 (b) and tRNA^Ser24 (c). (d) tRNA^Glu33 is shown as a representative example of a tRNA not cut by VapC-mt4 even though it contains the cleavage consensus sequence. In vitro-synthesized tRNAs 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 are indicated on the left. Note that in some cases extra bands are visible for the cleavage products because the T7 RNA polymerase used to synthesize the tRNAs frequently leads to 3′ end heterogeneity (usually ±1 nt)^61,62. Complete gel images for a–c are shown in Supplementary Fig. 3. Complete gel images for d along with full gels images of all 10 tRNAs that were weakly cut or not cut at all are shown in Supplementary Fig. 4.