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

Bullwinkle TJ, Reynolds NM, Raina M, Moghal A, Matsa E, Rajkovic A, Kayadibi H, Fazlollahi F, Ryan C, Howitz N, Faull KF, Lazazzera BA, Ibba M - Elife (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Ohio State University, Columbus, United States.

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.

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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.DOI:http://dx.doi.org/10.7554/eLife.02501.006
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fig2s2: 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.DOI:http://dx.doi.org/10.7554/eLife.02501.006

Mentions: Phenotypic microarrays (Biolog) were used to compare the growth of E. coli pheT(G318W) to wild type under 1920 growth conditions, and no significant changes were observed in the absence of PheRS editing. Additional experiments to investigate possible roles for editing under a range of other conditions, including heat shock, cold shock, pH stress and aging, failed to reveal differences compared to wild type. Growth of these strains was also compared in media containing varying concentrations of near-cognate p-Tyr in order to test the limits of EcPheRS specificity in the absence of post–transfer editing activity. Elevated concentrations of p-Tyr (>3 mM) did not affect the growth of E. coli pheT(G318W) compared to wild type (Figure 2A). Analysis of amino acid pools extracted from representative cells showed E. coli pheT(G318W) contained similar intracellular concentrations of p-Tyr and Phe as the wild type strain, indicating the pheT mutation has no effect on amino acid uptake (Table 1). In the absence of amino acid supplementation, the intracellular Phe:p-Tyr ratios were 1:1, and rose to 1:9 upon addition of p-Tyr. The growth of E. coli pheT(G318W) in the presence of m-Tyr, a non-proteinogenic amino acid previously shown to be a substrate for bacterial PheRS, was then investigated (Klipcan et al., 2009). Relative to wild type, growth of E. coli strain pheT(G318W) was inhibited in the presence of elevated intracellular concentrations of m-Tyr suggesting PheRS proofreading activity is needed to clear mischarged m-Tyr-tRNAPhe in vivo (Table 1; Figure 2B). Editing assays performed in vitro confirmed that, as with p-Tyr, post–transfer editing of m-Tyr-tRNAPhe by PheRS is ablated by the G318W mutation (Figure 2—figure supplement 1). The inhibitory effect of m-Tyr on growth in the absence of editing was also observed in E. coli mutants derived from strain MG1655 that, unlike the NP37 background, encodes an intact stringent response (Figure 2C). The pheT editing mutation was also constructed in the MG1655 background in order to confirm the m-Tyr growth phenotype was not specific to strains lacking the stringent response, where cells are unable to properly sense and respond to amino acid starvation. Growth of E. coli pheT(G318W) was also evaluated in the presence of ortho-tyrosine (o-Tyr) and 3,4-dihydroxy-L-phenylalanine (L-DOPA), oxidation products of Phe and p-Tyr, respectively (Maskos et al., 1992). Neither of these non-proteinogenic amino acids inhibited growth of wild type or the pheT(G318W) mutant E. coli strain (Figure 2—figure supplement 2).10.7554/eLife.02501.004Figure 2.Effect of non-cognate amino acids on the growth of editing deficient E. coli strains.


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

Bullwinkle TJ, Reynolds NM, Raina M, Moghal A, Matsa E, Rajkovic A, Kayadibi H, Fazlollahi F, Ryan C, Howitz N, Faull KF, Lazazzera BA, Ibba M - Elife (2014)

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.DOI:http://dx.doi.org/10.7554/eLife.02501.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2s2: 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.DOI:http://dx.doi.org/10.7554/eLife.02501.006
Mentions: Phenotypic microarrays (Biolog) were used to compare the growth of E. coli pheT(G318W) to wild type under 1920 growth conditions, and no significant changes were observed in the absence of PheRS editing. Additional experiments to investigate possible roles for editing under a range of other conditions, including heat shock, cold shock, pH stress and aging, failed to reveal differences compared to wild type. Growth of these strains was also compared in media containing varying concentrations of near-cognate p-Tyr in order to test the limits of EcPheRS specificity in the absence of post–transfer editing activity. Elevated concentrations of p-Tyr (>3 mM) did not affect the growth of E. coli pheT(G318W) compared to wild type (Figure 2A). Analysis of amino acid pools extracted from representative cells showed E. coli pheT(G318W) contained similar intracellular concentrations of p-Tyr and Phe as the wild type strain, indicating the pheT mutation has no effect on amino acid uptake (Table 1). In the absence of amino acid supplementation, the intracellular Phe:p-Tyr ratios were 1:1, and rose to 1:9 upon addition of p-Tyr. The growth of E. coli pheT(G318W) in the presence of m-Tyr, a non-proteinogenic amino acid previously shown to be a substrate for bacterial PheRS, was then investigated (Klipcan et al., 2009). Relative to wild type, growth of E. coli strain pheT(G318W) was inhibited in the presence of elevated intracellular concentrations of m-Tyr suggesting PheRS proofreading activity is needed to clear mischarged m-Tyr-tRNAPhe in vivo (Table 1; Figure 2B). Editing assays performed in vitro confirmed that, as with p-Tyr, post–transfer editing of m-Tyr-tRNAPhe by PheRS is ablated by the G318W mutation (Figure 2—figure supplement 1). The inhibitory effect of m-Tyr on growth in the absence of editing was also observed in E. coli mutants derived from strain MG1655 that, unlike the NP37 background, encodes an intact stringent response (Figure 2C). The pheT editing mutation was also constructed in the MG1655 background in order to confirm the m-Tyr growth phenotype was not specific to strains lacking the stringent response, where cells are unable to properly sense and respond to amino acid starvation. Growth of E. coli pheT(G318W) was also evaluated in the presence of ortho-tyrosine (o-Tyr) and 3,4-dihydroxy-L-phenylalanine (L-DOPA), oxidation products of Phe and p-Tyr, respectively (Maskos et al., 1992). Neither of these non-proteinogenic amino acids inhibited growth of wild type or the pheT(G318W) mutant E. coli strain (Figure 2—figure supplement 2).10.7554/eLife.02501.004Figure 2.Effect of non-cognate amino acids on the growth of editing deficient E. coli strains.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Ohio State University, Columbus, United States.

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.

Show MeSH
Related in: MedlinePlus