Some bacterial molecules have the social graces of certain 1700s French revolutionaries, or Shakespeare's Richard III, or the Queen of Hearts from Alice’s Adventures in Wonderland – in that all share a penchant for beheading those they encounter! In the microbial case, it's beheading of tRNAs.
Among the arsenal of toxins deployed in microbial turf squabbles are endoribonucleases that chop off the 3' ends of tRNAs. The resulting "headless" tRNAs can no longer carry amino acids and are no good for synthesizing proteins, to the great misfortune of the cell. These butcher-toxins are part of the CdiA family of proteins, a collection of destructive proteins used in contact-dependent growth inhibition. They come as a one-two punch: the aggressor cell secretes the CdiB/CdiA duo. The CdiB beta-barrel wedges into the victim's outer membrane to act as a backdoor to its cytoplasm. The CdiA effector partner cleaves its own C-terminus – the part of the protein that has teeth – and this CdiA-CT then slips through CdiB into the cell to do its cell-sabotaging thing, whether that is destroying the cell's DNA, slicing up rRNA, or very commonly, going after tRNAs with guillotine-esque purposes.
CdiA-CTs are selective going about their business. This is both in terms of which tRNA(s) are targeted, and what area on the tRNA is attacked, whether the anticodon or T-loop or – as highlighted here – the acceptor stem. Take a CdiA-CT toxin from enterohemorrhagic E. coli, for example, called EC869, which prefers Gln and Asn tRNAs and cleaves them between the 71st and 72nd nucleotides. CdiA-CTEC869 first commandeers the cell's own belongings: the two essential translation factors EF-Tu and EF-Ts, plus GTP. The catalytic efficiency of CdiA-CTEC869 soars when equipped with all three partners, forming a complex that then grabs onto its target tRNA. This broad overview has been known since 2017 from a study by Jones et al.
But how does the toxin sniff out its targets in the first place? A recent study by Wang et al. did some more fine-toothed biochemical probing into the process. First off, they noted that the tRNAs for Gln and Asn share a weak U1-A72 base pair at the top of the acceptor helix. But so do the tRNAs for Trp (here, a weak A1-U72 pair) and f-Met (here, a C1-A72 mismatch). Moreover, the Gln, Asn, and Trp tRNAs have a G73 as the discriminator base (the base that for many tRNAs is important in getting the correct amino acid attached). As one might suspect, CdiA-CTEC869 also chops up the Trp and f-Met tRNAs, suggesting that this flimsy tie at the ends of the tRNAs could be what dooms them. (Meanwhile, chopping-resistant tRNAAla has a G-C base pair there instead – that's more of a sailor's knot.) The toxin's recognition of its specific targets doesn't depend on the presence of an amino acid; in terms of slicing and dicing efficiency, it doesn't care whether its tRNAs are aminoacylated or not. What is important here is the presence of EF-Ts, which is required for the toxin complex to bind a tRNA (a pull-down experiment with the toxin not bound to EF-Ts yields no tRNAs!).
A note: there is a discrepancy between the 2017 and 2022 studies. Jones et al. 2017 found that EF-Tu is absolutely essential for CdiA-CTEC869 and that the toxin alone has no RNase activity, whereas Wang et al. 2022 found that toxin activity is still present at lower levels without EF-Tu, or EF-Ts, or GTP… Perhaps this just further highlights the pH-dependence of the toxin: the 2022 study tested for RNase activity in a pH range from 5.3 to 8.1, and found that dependence on EF-Tu/EF-Ts/GTP increased as pH increased. At low pH, there was barely any difference in activity between the toxin alone and the toxin in-complex, while at a higher pH, the toxin alone had almost no activity. Fittingly, the 2017 study did their assays around neutral pH. And, to account for the bit of 2022 solo-toxin activity at similar higher pH, perhaps there was a bit of contaminating EF-Tu or EF-Ts stuck to the purified toxin or tRNAs.
Finally, there is a region of CdiA-CTEC869 lined with several positively charged residues. This spot makes for a nice resting place (in both senses of the noun…) for the negatively charged RNAs. It is here, the authors propose, that the 3' end of the tRNA is held, and then chopped. Vive la révolution…