Fig. 6.

tRNA binding to Ntox28 nuclease domains. (A) Cross-linking of tRNA to CysK/CdiA-CT^EC536 complexes. E. coli cell lysates containing CysK and/or CdiA-CT(H178A)^EC536 were treated with formaldehyde, and cross-linked nucleoprotein complexes were purified by Ni²⁺-affinity chromatography. Purified samples were analyzed by SDS/PAGE (Top) and 50% urea PAGE (Bottom) to visualize proteins and nucleic acid, respectively. (B) CdiI^EC536 blocks tRNA cross-linking to CysK/CdiA-CT^EC536. E. coli cell lysates containing CysK-His₆, CysK-His₆/CdiA-CT(H178A)^EC536, and CysK-His₆/CdiA-CT(H178A)/CdiI^EC536 were treated with formaldehyde where indicated, followed by Ni²⁺-affinity chromatography, and gel analysis as described in A. (C) Tox28^Rlac interacts stably with tRNA substrate. Cell lysates containing His₆-Tox28(H114A)^Rlac and Imm^Rlac were treated as described above. To ascertain cross-linking specificity, EF-Tu-His₆ was also purified and analyzed for tRNA binding.