Response of E. coli to m-Tyrosine

To ensure faithful translation of the genetic code, some aminoacyl-tRNA synthetases have a quality control (QC) mechanism that allows them to remove similarly shaped, non-protein amino acids (NPAs) from a mischarged tRNA. In E. coli, the QC function of phenyalanyl-tRNA synthetase (PheRS) is required for resistantce to the toxic NPA, meta-Tyrosine (m-Tyr).  Here, we sought to understand the mechanism of m-Tyr toxicity. Using RNA-seq, we observed >500 genes were differential expressed after the addition of m-Tyr. The most strongly up-regulated genes are involved in unfolded-protein stress response, and cells exposed to m-Tyr contained large, electron-dense protein aggregates, indicating that m-Tyr destabilized a large fraction of the proteome. Additionally, we observed that amino acid biosynthesis and transport regulons, controlled by ArgR, TrpR, and TyrR, and the stringent-response regulon, controlled by DksA/ppGpp, were differentially expressed. m-Tyr resistant mutants were isolated and found to have altered a promoter to increase expression of the enzymes for Phe production or to have altered transporters, which likely result in less uptake or increased efflux of m-Tyr. These findings indicate that QC by PheRS is the critical point for resisting the toxic effects of m-Tyr, when m-Tyr has passed the QC checkpoint and enters the proteome, it is sequestered in protein aggregates that are eventually lost through cell division. A 5 ml LB culture was inoculated from a single colony of BAL4703 (pheT(G318W)) strain and grown until it reached mid-log phase (OD600 0.4-0.6). A 1 ml sample of the cells were then washed in 0.9% NaCl and used to inoculate a 100 ml, M9 minimal media culture in a 500 ml flask to an OD600 of 0.001. The M9 culture was allowed to grow with shaking at 37°C until it reached an OD600 of 0.5. A sample of cells was taken at this time (t=0) for RNA purification and CFU/ml determination. A m-Tyr solution was then added to the culture to 0.5 mM. The culture was then allowed to continue growing at 37C with shaking. Additional sampling for RNA purification and CFU/ml measurements was done at 30, 60, and 120 minutes after m-Tyr addition. This protocol was repeated for a total of three biological replicates.