ABSTRACT

Lamotrigine (Ltg), an anticonvulsant drug, targets initiation factor 2 (IF2), compromises ribosome biogenesis and causes toxicity to Escherichia coli. However, our understanding of Ltg toxicity in E. coli remains unclear. While our in vitro assays reveal no effects of Ltg on the ribosome-dependent GTPase activity of IF2 or its role in initiation as measured by dipeptide formation in a fast kinetics assay, the in vivo experiments show that Ltg causes accumulation of the 17S precursor of 16S rRNA and leads to a decrease in polysome levels in E. coli. IF2 overexpression in E. coli increases Ltg toxicity. However, the overexpression of initiator tRNA (i-tRNA) protects it from the Ltg toxicity. The depletion of i-tRNA or overexpression of its 3GC mutant (lacking the characteristic 3GC base pairs in anticodon stem) enhances Ltg toxicity, and this enhancement in toxicity is synthetic with IF2 overexpression. The Ltg treatment itself causes a detectable increase in IF2 levels in E. coli and allows initiation with an elongator tRNA, suggesting compromise in the fidelity/specificity of IF2 function. Also, Ltg causes increased accumulation of ribosome-binding factor A (RbfA) on 30S ribosomal subunit. Based on our genetic and biochemical investigations, we show that Ltg compromises the function of i-tRNA/IF2 complex in ribosome maturation.

摘要

拉莫三嗪 (Ltg) 是一种抗惊厥药物，以起始因子 2 (IF2) 为靶标，损害核糖体生物发生并对大肠杆菌产生毒性。然而，我们对 Ltg 在大肠杆菌中的毒性的理解仍不清楚。虽然我们的体外试验表明，Ltg 对 IF2 的核糖体依赖性 GTP 酶活性或其在快速动力学试验中通过二肽形成测量的启动作用没有影响，但体内实验表明，Ltg 会导致 16S 的 17S 前体积累rRNA 并导致大肠杆菌中多核糖体水平下降。大肠杆菌中 IF2 过度表达会增加 Ltg 毒性。然而，起始子 tRNA (i-tRNA) 的过度表达可保护其免受 Ltg 毒性。i-tRNA 的耗尽或其 3GC 突变体（缺乏反密码子干中特征性 3GC 碱基对）的过度表达增强了 Ltg 毒性，并且这种毒性增强是由 IF2 过度表达合成的。Ltg 处理本身会导致大肠杆菌中 IF2 水平可检测到的增加，并允许使用延伸 tRNA 启动，这表明 IF2 功能的保真度/特异性受到损害。此外，Ltg 会导致 30S 核糖体亚基上核糖体结合因子 A (RbfA) 的积累增加。基于我们的遗传和生化研究，我们发现 Ltg 会损害 i-tRNA/IF2 复合物在核糖体成熟中的功能。