Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA–CT) into neighboring bacteria. The CdiA–CT of enterohemorrhagic Escherichia coli EC869 (CdiA–CT(EC869)) cleaves the 3′-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA–CT(EC869) has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA–CT(EC869). Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA–CT(EC869) to the same extent in the presence of Tu/Ts, and the CdiA–CT(EC869):Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA–CT(EC869) interacts with domain II of Tu, thereby preventing the 3′-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA–CT(EC869):Tu structure suggests that the 3′-portion of tRNA relocates into the CdiA–CT(EC869) active site, located on the opposite side to the CdiA–CT(EC869) :Tu interface, for tRNA cleavage. Thus, CdiA–CT(EC869) is recruited to Tu:GTP:Ts, and CdiA–CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA–CT(EC869) for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA–CT(EC869).