Fig. 5.

R. lactaris Tox28 is a CysK-independent tRNase. (A) CdiA-CT/CdiI^EC536-DAS and Tox/Imm^Rlac-DAS expression constructs were introduced into E. coli cysK⁺ and ∆cysK cells, and transformants were selected on media supplemented with glucose or arabinose. (B) Protein binding assays. The indicated proteins were mixed and subjected to Ni²⁺-affinity chromatography. The free lanes represent proteins that failed to bind the affinity matrix, and the bound lanes show proteins eluted with imidazole. (C) In vitro nuclease assays. Purified CdiA-CT^EC536 or Tox28^Rlac was incubated with total cellular RNA. Reactions were supplemented with purified CysK, CdiI^EC536, or Imm^Rlac where indicated.