Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code

Abstract

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

Keywords: E. coli; S. cerevisiae; biochemistry; infectious disease; microbiology; protein synthesis; stress; translation.

Figure 1.. Chromosomal editing mutants of E. coli and S. cerevisiae.

(A) Post–transfer hydrolysis of [¹⁴C]- Tyr-tRNA^Phe (1 µM) by cell-free extracts isolated from wild type (●) and pheT(G318W) (■) E. coli strains (140 mg/ml total protein concentration) or buffer (▲) at 37°C. (B) Posttransfer editing activity of βD243A ctPheRS in S. cerevisiae. Reactions were performed at 37°C with 2 μM Tyr-tRNA^Phe and S. cerevisiae wild type FRS1 or frs1-1 (D243A) cell-free extracts normalized to aminoacylation activity (Reynolds et al., 2010). Data points are the mean of at least three independent experiments, with errors bars representing ±1 SD. DOI: http://dx.doi.org/10.7554/eLife.02501.003

Figure 2.. Effect of non-cognate amino acids on the growth of editing deficient E. coli strains.

Growth of E. coli pheT(G318W) strain (grey bars) relative to wild type (black bars) under increasing concentrations of L-p-Tyr (A) or D,L-m-Tyr (B) relative to Phe. Cultures were grown in M9 minimal media supplemented with amino acids expressed as a ratio of Phe:Tyr. A ratio of 1:1 corresponds to 3 µM of each amino acid. (C) Growth of PheRS editing deficient strain of E. coli in an MG1655 background in the presence of different tyrosine isomers at 37°C. Bars are the mean of three independent cultures, with errors bars representing ± SD. DOI: http://dx.doi.org/10.7554/eLife.02501.004

Figure 2—figure supplement 1.. EcPheRS post–transfer editing of mischarged tRNA^Phe substrates.

Hydrolysis of 0.1 µM E. coli p-Tyr-[³²P]-tRNA^Phe (dashed lines) or m-Tyr-[³²P]-tRNA^Phe (solid lines) in the presence of 10 nM wild type EcPheRS (■) G318W EcPheRS (=) or buffer (▲) at 37°C. Data points are the mean of three independent experiments, with errors bars representing ± SD. DOI: http://dx.doi.org/10.7554/eLife.02501.005

Figure 2—figure supplement 2.. E. coli PheRS editing requirement for tyrosine isomers.

Growth of PheRS editing deficient E. coli at 37°C after 16 hr in M9 minimal media supplemented with increasing concentrations of (A) o-Tyr or (B) L-dopa. (C) Aminoacylation of [³²P]-tRNA^Phe with o-Tyr (=) or L-dopa (■) by E. coli G318W PheRS (1 µM). Bars are the mean of three independent cultures, with errors bars representing ± SD. DOI: http://dx.doi.org/10.7554/eLife.02501.006

Figure 3.. Effect of non-cognate amino acids on the growth of an editing deficient S. cerevisiae strain.

Growth of yeast frs1-1 (D243A) strain (grey bars) relative to a wild type strain (black bars) under increasing concentrations of L-p-Tyr (A) or D,L-m-Tyr (B) relative to Phe. Cultures were grown in minimal media supplemented with amino acids expressed as a ratio of Phe:Tyr. A ratio of 1:1 corresponds to 3 µM of each amino acid. Data points are the mean of three independent cultures, with errors bars representing ±1 SD. DOI: http://dx.doi.org/10.7554/eLife.02501.008

Figure 4.. Tyrosine isomers as substrates for tRNA^Phe aminoacylation by PheRS variants.

tRNA^Phe aminoacylation activities of (A) wild type and (B) G318W E. coli PheRS for 60 μM cognate Phe and non-cognate p- and m-Tyr substrates. Aminoacylation activities of (C) wild type cytoplasmic and (D) wild type mitochondrial S. cerevisiae PheRS for 100 μM cognate Phe and non-cognate p- and m-Tyr substrates. Data points are the mean of three independent experiments, with errors bars representing ± SD. DOI: http://dx.doi.org/10.7554/eLife.02501.009

Figure 5.. ScctPheRS post–transfer editing of mischarged tRNA^Phe substrates.

Hydrolysis of 0.1 µM yeast (A) p-Tyr-[³²P]-tRNA^Phe or (B) m-Tyr-[³²P]-tRNA^Phe in the presence of 10 nM wild type ScctPheRS (●) D243A ScctPheRS (■) or buffer (▲) at 37°C. Data points are the mean of three independent experiments, with errors bars representing ± SD. DOI: http://dx.doi.org/10.7554/eLife.02501.011

Figure 6.. Incorporation of m-Tyr into the proteome of E. coli.

(A) In vitro 70S ribosomal di-peptide synthesis with either Phe-tRNA^Phe or m-Tyr-tRNA^Phe (B) LC-MS/MS-MRM quantification of m-Tyr and Phe in protein hydrolysis isolated from E. coli expressed as molar ratio of m-Tyr to Phe. Wild type (Wt) and pheT(G318W) strains grown in M9 minimal media alone and supplemented with m-Tyr are shown. Error bars represent ± standard error of means. DOI: http://dx.doi.org/10.7554/eLife.02501.012

Figure 6—figure supplement 1.. p-Tyr is not misincorported in the proteome of E. coli at Phe codons.

Mass spectroscopy quantification of p-Tyr and Phe in protein hydrolysis isolated from E. coli expressed as molar ratio of p-tyr to Phe. Wild type and pheT(G318W) strains grown in in M9 minimal media alone and supplemented with p-Tyr are shown. Error bars represent ± standard error of means. DOI: http://dx.doi.org/10.7554/eLife.02501.013

Figure 6—figure supplement 2.. E. coli TyrRS uses m-Tyr.

Aminoacylation of E. coli [³²P]-tRNA^Phe transcript (0.5 μM) with m-Tyr (1 mM) by E. coli TyrRS (50 nM) at 25°C. DOI: http://dx.doi.org/10.7554/eLife.02501.014

Figure 7.. Requirement for PheRS posttransfer editing in ROS conditions in vivo.

(A) LC-MS/MS-MRM chromatograms for p-, m- and o-Tyr (m/z 182→136 transition) extracted from cells grown in the absence (left) and presence (right) of H₂O₂ and FeSO₄. (B) Growth of E. coli pheT(G318W) strain relative to wild type in M9 minimal media supplemented with 0.1 mM FeSO₄ and increasing concentrations of H₂O_2. Bars are the mean of three independent cultures, with errors bars representing ± SD. DOI: http://dx.doi.org/10.7554/eLife.02501.015