Oxidation of Cellular Amino Acid Pools Leads to Cytotoxic Mistranslation of the Genetic Code

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

The Nature of meta-Tyrosine Toxicity to Phenylalanyl-tRNA Synthetase Editing-Defective Escherichia coli

Faithful translation of the genetic code into amino acid sequences is important for the viability of organisms. One source of error in translation is the mischarging of tRNAs with the incorrect amino acid due to structural similarities between the cognate and non-cognate amino acids. If gone unchecked, these mischarged tRNAs would provide an amino acid to the ribosome that does not match its codon, thereby causing mistranslation of the mRNA sequence. The proteins that are responsible for charging tRNAs with the correct amino acids are called aminoacyl-tRNA synthetases (aaRS). Some of these aaRSs have evolved an editing mechanism that allows them to cleave off a non-cognate amino acid from the mischarged tRNA, which is broadly conserved across all domains of life. This editing activity seems like it would be essential for life, however there are many examples of organisms who have lost their editing function to no ill effect. Moreover, there are examples of organisms that have conserved their editing function, but do not show a growth defect when it is eliminated, such as E. coli and its phenylalanine aaRS (PheRS). We chose to study E. coli’s PheRS to understand why its editing function is evolutionarily conserved. We discovered that the non-protein amino acid meta-Tyrosine (m-Tyr) is toxic to PheRS editing-defective (PheRS edit-) E. coli. We then sought to understand why m-Tyr is so toxic to PheRS edit- cells. We used chemical mutagenesis to find m-Tyr resistant mutants and then performed whole genome sequencing to find mutated genes that could contribute to the resistance. We found that mutations in uptake and efflux transport could provide resistance by keeping or getting m-Tyr out of the cell. We also identified a resistance mutation that likely elevated Phe production, which provided resistance by most likely increasing competitive inhibition of the m-Tyr. We also observed PheRS edit- E. coli after m-Tyr exposure directly via light and electron microscopy. We observed large protein aggregates forming in the cells, which indicated that the m-Tyr destabilized a large fraction of the proteome. We also performed transcriptomic analysis of PheRS edit- E. coli after m-Tyr exposure to see what stress responses they used to deal with m-Tyr toxicity. We found a strong induction of the unfolded protein stress response, as well as oxidative stress, DNA damage stress, and indications of lost ion homeostasis. Based on these findings, we proposed a model of m-Tyr toxicity that involves a cascading and self-reinforcing chain reaction of cellular stresses that ultimately leads to cell death

Synthetic Homoserine Lactone Sensors for Gram-Positive Bacillus subtilis Using LuxR-Type Regulators

A universal biochemical signal for bacterial cell–cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that Gram-negative and Gram-positive bacteria generally communicate via homoserine lactones (HSLs) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for Gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (−35, −16, −10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using the statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence–function relationships for synthetic promoter design. We present the first known set of functional HSL sensors (≥20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p-coumaroyl-HSL, 3-oxohexanoyl-HSL, n-butyryl-HSL, and n-(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a Gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia

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

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

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

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

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

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