1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
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Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code

  1. Tammy J Bullwinkle
  2. Noah M Reynolds
  3. Medha Raina
  4. Adil Moghal
  5. Eleftheria Matsa
  6. Andrei Rajkovic
  7. Huseyin Kayadibi
  8. Farbod Fazlollahi
  9. Christopher Ryan
  10. Nathaniel Howitz
  11. Kym F Faull
  12. Beth A Lazazzera
  13. Michael Ibba  Is a corresponding author
  1. Ohio State University, United States
  2. Center for RNA Biology, Ohio State University, United States
  3. Semel Institute for Neuroscience and Human Behavior, University of California, United States
  4. University of California, Los Angeles, United States
Research Article
Cite this article as: eLife 2014;3:e02501 doi: 10.7554/eLife.02501
7 figures and 2 tables

Figures

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

(A) Post–transfer hydrolysis of [14C]- Tyr-tRNAPhe (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-tRNAPhe 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.

https://doi.org/10.7554/eLife.02501.003
Figure 2 with 2 supplements
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.

https://doi.org/10.7554/eLife.02501.004
Figure 2—figure supplement 1
EcPheRS post–transfer editing of mischarged tRNAPhe substrates.

Hydrolysis of 0.1 µM E. coli p-Tyr-[32P]-tRNAPhe (dashed lines) or m-Tyr-[32P]-tRNAPhe (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.

https://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]-tRNAPhe 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.

https://doi.org/10.7554/eLife.02501.006
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.

https://doi.org/10.7554/eLife.02501.008
Tyrosine isomers as substrates for tRNAPhe aminoacylation by PheRS variants.

tRNAPhe 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.

https://doi.org/10.7554/eLife.02501.009
ScctPheRS post–transfer editing of mischarged tRNAPhe substrates.

Hydrolysis of 0.1 µM yeast (A) p-Tyr-[32P]-tRNAPhe or (B) m-Tyr-[32P]-tRNAPhe 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.

https://doi.org/10.7554/eLife.02501.011
Figure 6 with 2 supplements
Incorporation of m-Tyr into the proteome of E. coli.

(A) In vitro 70S ribosomal di-peptide synthesis with either Phe-tRNAPhe or m-Tyr-tRNAPhe (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.

https://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.

https://doi.org/10.7554/eLife.02501.013
Figure 6—figure supplement 2
E. coli TyrRS uses m-Tyr.

Aminoacylation of E. coli [32P]-tRNAPhe transcript (0.5 μM) with m-Tyr (1 mM) by E. coli TyrRS (50 nM) at 25°C.

https://doi.org/10.7554/eLife.02501.014
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 H2O2. Bars are the mean of three independent cultures, with errors bars representing ± SD.

https://doi.org/10.7554/eLife.02501.015

Tables

Table 1

Amino acid pools in wild type and editing defective E. coli strains

https://doi.org/10.7554/eLife.02501.007
StrainSupplementm-Tyr (µM)*p-Tyr (µM)Phe (µM)p-Tyr/Phem-Tyr/Phe
Wild type+ m-Tyr2.9 ± 0.060.56 ± 0.10.63 ± 0.20.9 ± 0.05 ± 1
pheT(G318W)+ m-Tyr2.7 ± 0.50.46 ± 0.020.90 ± 0.20.9 ± 0.26 ± 1
Wild type+ p-TyrND11 ± 40.91 ± 0.112 ± 4ND
pheT(G318W)+ p-TyrND8.9 ± 0.40.93 ± 0.19.7 ± 1ND
  1. *

    Concentrations of intracellular Phe and Tyr isomers isolated from wild type and pheT(G318W) E. coli strains grown in M9 minimal media supplemented with either m-Tyr or p-Tyr.

  2. ND indicates concentrations were below the detectable limit (0.01 µM).

Table 2

Steady-state kinetic constants for amino acid activation by PheRS from E. coli and S. cerevisiae cytoplasmic PheRS

https://doi.org/10.7554/eLife.02501.010
Phem-Tyrp-TyrSpecificity (kcat/KM/kcat/KM)
PheRSKM (µM)kcat (s−1)kcat/KM (s−1/µM)KM (µM)kcat (s−1)kcat/KM (s−1/µM)kcat/KM (s−1/µM)Phe/m-TyrPhe/p-Tyr
E. coli18 ± 45.2 ± 20.29247 ± 602.1 ± 0.80.0081.1 × 10-4352650
Yeast ct16 ± 226 ± 41.61150 ± 23026 ± 40.0230.01471120

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