Structural basis for 16S ribosomal RNA cleavage by the cytotoxic domain of colicin E3

The vital role of the ribosome in translating genetic information into proteins makes it an obvious target for various natural antibiotics and bacteriocins—protein toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. Recent progress in ribosome crystallography has provided an enormous amount of structural detail, helping to explain how antibiotics interfere with protein translation1–4. However, questions on the specific action of bacteriocins await a high-resolution structure of their complexes with ribosomes. Bacteriocins that target the ribosome include colicins E3, E4 and E6, as well as cloacin DF13, which all show specific 16S rRNase activity5,6. Colicin E3 is a plasmid-encoded 60-kDa protein that is produced during times of stress by some strains of Escherichia coli. It contains three domains that are required for the toxin to enter and kill the cell: the N-terminal translocation domain (T), the central receptor-binding domain (R) and the C-terminal cytotoxic domain7. Together with E3, the cell coexpresses and releases an immunity protein (Im3) that binds with high affinity (picomolar Kd) to the E3 cytotoxic domain and prevents the producing cell from committing suicide by specifically inactivating the RNase activity8–10. The E3–Im3 complex is expressed and released from cells through the SOS response, penetrating a target cell through interactions of the R and T domains with specific receptors and translocators in the outer membrane of E. coli. During the translocation process the Im3 protein dissociates from the complex, leaving the E3-rRNase to exert its cytotoxic activity11. E3-rRNase cleaves a single phosphodiester bond between A1493 and G1494 (Escherichia coli numbering, used throughout the manuscript) in the decoding site (A site) in helix 44 of ribosomal 16S rRNA12,13, consequently impairing protein translation and eventually causing cell death. Biochemical and structural studies on colicin E3 have identified many residues implicated in the catalytic activity of the rRNase, including residues Arg40, Arg42, Asp55, His58, Glu60, Glu62 and Arg90 (refs. 14–17). Furthermore, in vitro assays have shown that ribosomes with E3-cleaved 16S rRNA in the A site show impaired decoding defects, resulting in the production of short peptides18. However, despite this plethora of biochemical and structural data, the nature of the high specificity of E3 16S rRNase, its cleavage mechanism and the structural and biological consequences of 16S RNA cleavage remain speculative and unresolved. To address these questions at the atomic level, we solved to 3.2-Å resolution the crystal structure of E3-rRNase bound to Thermus thermophilus 70S ribosomes in complex with mRNA and tRNAs in the post-cleavage state. The structure provides new insights into bacteriocin action on ribosomes.