Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

Abstract

Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli. This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.

Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome

“…Lysogeny broth (LB) was used for all growth experiments in rich media. M9 media was prepared for the minimal media experiment supplemented with exogenous serine [ 13 ]. SOB and SOC media were used for CRISPR/Cas9-mediated strain engineering.…”

“…The AlaRS C666A suppressor in alaS at nucleotide position G1651T (encoding AlaRS D551Y) was made in an isogenic MG1655 strain using the “gene gorging” method as has previously been described [ 13 ]. Suppressor mutations encoding substitutions at R561 were also generated in isogenic MG1655 derivative strains.…”

“…The construction and utility of the pLK-Amp S68A mistranslation reporter have been previously described [ 13 ]. In brief, β-lactamase, a gene product used for conferring ampicillin resistance, contains an essential serine residue at position S68.…”

“…While considerable work has been devoted to characterizing the enzymatic activity of aaRSs, the exploration of the role of these enzymes in vivo has only been recently investigated [ 10 , 11 , 12 , 13 ]. It was previously observed that an alanine substitution of an essential cysteine in the AlaRS proofreading domain leads to mis-aminoacylation of serine on tRNA Ala in vitro [ 9 ].…”

“…It was previously observed that an alanine substitution of an essential cysteine in the AlaRS proofreading domain leads to mis-aminoacylation of serine on tRNA Ala in vitro [ 9 ]. E. coli with the same substitution (AlaRS C666A) show defects in maintaining proteome homeostasis in E. coli [ 13 ]. To expand on these efforts, we sought to identify suppressors of the AlaRS C666A phenotype in hopes of further characterizing the mechanisms by which AlaRS fidelity promotes cellular homeostasis.…”