Antimicrobial activity of fusidic acid in Escherichia coli is dependent on the relative levels of ribosome recycling factor and elongation factor G

During protein synthesis, elongation factor G (EFG) participates at the steps of translocation and ribosome recycling. Fusidic acid (FA) is a bacteriostatic antibiotic, which traps EFG on ribosomes, stalling them on mRNAs. How the bacterial susceptibility to FA is determined, and which of the two functions of EFG (translocation or ribosome recycling) is more vulnerable, has remained debatable. The in vivo studies addressing these aspects of FA mediated inhibition of protein synthesis are lacking. Here, we used a system of Escherichia coli strains and their complementation/supplementation with the plasmid borne copies of the inducible versions of EFG and ribosome recycling factor (RRF) genes. Additionally, we investigated FA sensitivity in a strain with increased proportion of stalled ribosomes. We show that the cells with high EFG/RRF (or low RRF/EFG) ratios are more susceptible to FA than those with low EFG/RRF (or high RRF/EFG) ratios. Our in vivo observations are consistent with the recent in vitro reports of dependence of FA susceptibility on EFG/RRF ratios, and the notion that an overriding target of FA is the translocation function of EFG. An applied outcome of our in vivo study is that FA mediated growth inhibition could be facilitated by depletion or inactivation of cellular RRF

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

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

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas

<p>Initiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the <i>Escherichia coli</i> model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.</p