Substrate recognition mechanism of tRNA-targeting ribonuclease, colicin D, and an insight into tRNA cleavage-mediated translation impairment.

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNA ^Arg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNA ^Arg s; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNA ^Arg s from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNA ^Arg _ICG , the most abundant species of E. coli tRNA ^Arg s, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNA ^Arg _ICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNA ^Arg _ICG dominant-negatively impairs translation in vitro . Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNA ^Arg _ICG , which is consistent with our structural model. Finally, elevation of cleaved tRNA ^Arg _ICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNA ^Arg _ICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops. Abbreviations: mnm ⁵ U: 5-methylaminomethyluridine; mcm ⁵ s ² U: 5-methoxycarbonylmethyl-2-thiouridine.