Lamotrigine compromises the fidelity of initiator tRNA recruitment to the ribosomal P-site by IF2 and the RbfA release from 30S ribosomes in Escherichia coli

RNA Biol. 2023 Jan;20(1):681-692. doi: 10.1080/15476286.2023.2253395.

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.

Keywords: Ltg; Ribosome; initiation factor 2; initiator tRNA; lamotrigine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anticonvulsants*
  • Escherichia coli / genetics
  • Escherichia coli Proteins* / genetics
  • Lamotrigine / pharmacology
  • Prokaryotic Initiation Factor-2
  • RNA, Ribosomal, 16S / genetics
  • RNA, Transfer, Met / genetics
  • Ribosomal Proteins
  • Ribosomes

Substances

  • Lamotrigine
  • Anticonvulsants
  • Prokaryotic Initiation Factor-2
  • RNA, Transfer, Met
  • RNA, Ribosomal, 16S
  • RbfA protein, E coli
  • Ribosomal Proteins
  • Escherichia coli Proteins

Grants and funding

This work was supported by grants from the Science and Engineering Research Board [EMR/2016/005617], the Department of Biotechnology [BT/PR28058, and BT/PR13522], Jamshedji Tata Trust, India [R(HR)Tata Chairs/2020-854/78] to UV and the Swedish Research Council (2018-05419, 2016-06264, and 2018-05498) to SS. UV is a J. N. Tata Chair Professor and a J.C. Bose fellow (SERB). Sudhir Singh is a DBT-RA supported by the DBT-RA Program in Biotechnology and Life Sciences to carry out this work. The authors acknowledge the DBT-IISc partnership programme [BT/PR27952], and the Centre of Advanced Studies (UGC), the DST-FIST level II infrastructure support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.