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

摘要

Colicin D是一种质粒编码的细菌素，可特异性切割敏感大肠杆菌细胞的tRNA ^Arg。大肠杆菌具有四个同等接受的tRNA ^Arg。切割发生在反密码子环的3'端，导致敏感细胞的翻译受损。tRNA形成共同的L形结构，并具有许多保守的核苷酸，限制了tRNA身份元件。因此，大肠菌素D如何从敏感大肠杆菌细胞的tRNA库中选择tRNA ^Arg的方法很有趣。在这里，我们通过生化分析和结构建模揭示了大肠菌素D的识别机制。Colicin D识别tRNA ^Arg _ICG，tRNA ^Arg _ICG是 大肠杆菌tRNA ^Arg处于其反密码子环和D臂，并通过将其反密码子环序列与其他序列区分开来将其选择为最优选的底物。已经假定翻译损伤是由于切割引起的完整tRNA分子的减少引起的。然而，我们发现完整的tRNA ^Arg _ICG的细胞内水平并不能确定此类切割后敏感细胞的活力。而是分裂的积累。切割的tRNA ^Arg _ICG显着不利于体外翻译。此外，我们发现EF-Tu是tRNA递送所必需的，它不与大肠菌素D竞争结合tRNA ^Arg _ICG，这与我们的结构模型一致。最后，裂解的tRNA ^Arg _ICG水平升高会降低敏感细胞的活力。这些结果表明，切割的tRNA ^Arg _ICG以EF-Tu依赖性方式瞬时占据核糖体A位点，导致翻译损伤。该策略也应适用于其他靶向tRNA的RNase，因为它们也可以识别反密码子环。

缩写： mnm ⁵ U：5-甲基氨基甲基尿苷；m = ⁵。mcm ⁵ s ² U：5-甲氧羰基甲基-2-硫尿苷