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

ABSTRACT: Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNA^Arg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNA^Args; 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^Args 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^Args, 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: mnm⁵U: 5-methylaminomethyluridine; mcm⁵s²U: 5-methoxycarbonylmethyl-2-thiouridine.

SUBMITTER: Ogawa T 

PROVIDER: S-EPMC8244772 | biostudies-literature |

REPOSITORIES: biostudies-literature