Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae

Abstract

Amino acid starvation activates the protein kinase Gcn2p, leading to changes in gene expression and translation. Gcn2p is activated by deacylated tRNA, which accumulates when tRNA aminoacylation is limited by lack of substrates or inhibition of synthesis. Pairing of amino acids and deacylated tRNAs is catalyzed by aminoacyl-tRNA synthetases, which use quality control pathways to maintain substrate specificity. Phenylalanyl-tRNA synthetase (PheRS) maintains specificity via an editing pathway that targets non-cognate Tyr-tRNAPhe. While the primary role of aaRS editing is to prevent misaminoacylation, we demonstrate editing of misaminoacylated tRNA is also required for detection of amino acid starvation by Gcn2p. Ablation of PheRS editing caused accumulation of Tyr-tRNAPhe (5%), but not deacylated tRNAPhe during amino acid starvation, limiting Gcn2p kinase activity and suppressing Gcn4p-dependent gene expression. While the PheRS-editing ablated strain grew 50% slower and displayed a 27-fold increase in the rate of mistranslation of Phe codons as Tyr compared to wild type, the increase in mistranslation was insufficient to activate an unfolded protein stress response. These findings show that during amino acid starvation a primary role of aaRS quality control is to help the cell mount an effective stress response, independent of the role of editing in maintaining translational accuracy.

Regulation of Sensing, Transportation, and Catabolism of Nitrogen Sources in Saccharomyces cerevisiae

“…Aminoacyl-tRNA synthetases catalyze the reaction of amino acids with cognate uncharged tRNAs. Their proofreading function is essential for minimizing mistranslation through hydrolysis of misactivated aminoacyl adenylates (pretransfer editing) and hydrolysis of misaminoacylated aa-tRNA (posttransfer editing) (186). The absence of editing causes accumulation of misaminoacylated tRNA, but not deacylated tRNA, under amino acid starvation conditions, which represses the transcription of Gcn2 (186).…”

“…Their proofreading function is essential for minimizing mistranslation through hydrolysis of misactivated aminoacyl adenylates (pretransfer editing) and hydrolysis of misaminoacylated aa-tRNA (posttransfer editing) (186). The absence of editing causes accumulation of misaminoacylated tRNA, but not deacylated tRNA, under amino acid starvation conditions, which represses the transcription of Gcn2 (186). The activation of Gcn2 induces the phosphorylation of Ser51 of eIF2␣ (187)(188)(189)(190)(191) to enhance its affinity for the GEF eIF2B, which inhibits the exchange rate from the GDP-to GTP-bound status of eIF2 and finally reduces the TC formation rate (33,192).…”

Loss of protein synthesis quality control in host-restricted organisms

“…The question still remains: How do so many intracellular pathogens and endosymbiotic bacteria survive with defects in the editing domains? These domains have been long considered a highly conserved and essential system for accurate protein synthesis: mutations in these regions were shown to cause protein misfolding and genetic disorders in mammals (27)(28)(29)(30)(31); an impaired amino acid-starvation response in yeast (32); and growth defects, cell death, hypersensitivity to elevated amino acid levels, and mistranslation of up to 10% of codons that correspond to a defective synthetase in E. coli (33,34).…”

Proteomics Analysis for Amino Acid Misincorporation Detection: Mini Review

“…Several mistranslation errors were identified using this approach in different organisms and cell lines, namely CHO cells, which are frequently used for recombinant expression of target proteins [44,[46][47][48][49]. This strategy was also used to demonstrate that the tyrosyl-tRNA synthetase is highly error prone [50,51] and that the editing site of some aminoacyl-tRNA synthetases controls the sensitivity of amino acid stress responses [52].…”