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

ABSTRACT

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-CTEC869) 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-CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and TuTs complex promotes tRNA cleavage by CdiA-CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA-CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA-CTEC869TuTstRNA(aa-tRNA) complex formed in the presence of GTP. CdiA-CTEC869 interacts with domain II of Tu, thereby preventing the 3'-moiety of tRNA to bind to Tu as in canonical TuGTPaa-tRNA complexes. Superimposition of the TuGTPaa-tRNA structure onto the CdiA-CTEC869Tu structure suggests that the 3'-portion of tRNA relocates into the CdiA-CTEC869 active site, located on the opposite side to the CdiA-CTEC869Tu interface, for tRNA cleavage. Thus, CdiA-CTEC869 is recruited to TuGTPTs, and CdiA-CTTuGTPTs recognizes substrate tRNAs and cleaves them. TuGTPTs serves as a reaction scaffold that increases the affinity of CdiA-CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA-CTEC869.