## 3 Results
### AlaRS C666A Suppressors Do Not Prevent Serine Mistranslation  
To directly observe the potential for Ala to Ser mistranslation in the suppressor strains, a recently described mistranslation reporter [13] was applied to all of the single and double mutants. The mistranslation reporter utilizes a mutated \(\beta\)-lactamase gene in which an essential serine residue has been mutated to encode an alanine [31]. Under conditions of high AlaRS fidelity, the mutant \(\beta\)-lactamase product will be translated and the cells will be unable to grow on \(\beta\)-lactam antibiotics (e.g., ampicillin). If Ala to Ser mistranslation is occurring, the wildtype \(\beta\)-lactamase protein will be made and the cells will grow in the presence of the antibiotic. This reporter has recently been used to show that in the AlaRS C666A variant strain, Ala to Ser mistranslation is occurring in vivo [13]. Upon transformation of the reporter and plating of the suppressor strains, all variants that contained the C666A allele were able to grow on ampicillin (Figure 3). This observation suggests that the C666A substitution is sufficient to cause protein mistranslation even in the presence of additional second-site suppressors. None of the single AlaRS suppressor variants were able to grow in the presence of antibiotics suggesting that these substitutions alone do not negatively affect the hydrolytic proofreading activity of these enzymes in vivo. One limitation of this qualitative assay is the inability to discern quantifiable changes in levels of mistranslated proteins. It is possible that despite the accumulation  
Figure 2: Suppressor mutants alleviate AlaRS C666A-associated growth defects. Suppressor mutants alleviated the AlaRS C666A growth defects in (**A**) LB (Lysogeny broth) and (**B**) M9 minimal media supplemented with non-cognate serine. of mistranslated proteins in the second-site suppressors, the levels of mistranslation are still reduced compared to the single AlaRS C666A variant. Phenotypes Associated with AlaRS-Mediated Proteome Dysregulation Are Alleviated in with the Second-Site Suppressor Mutations  
Previous efforts studying the effect of AlaRS mistranslation in _E. coli_ identified that not only was protein mistranslation occurring, but the accumulation of mistranslated proteins led to dysregulation of the _E. coli_ proteome [13]. This observation suggested that regardless of the individual protein that may contain a misincorporated amino acid, the Ala to Ser substitution itself was not tolerated globally and caused activation of several stress response cascades that could be reproducibly observed with population-level analyses. The first indication that protein regulation may be altered in the _E. coli_ AlaRS strain was the observation that in the absence of AlaRS proofreading, _E. coli_ exhibited an accentuated slow growth phenotype when grown in rich media at 42 \({}^{\circ}\)C. The delayed growth response to the heat stress potentially suggested that these AlaRS proofreading-deficient cells were unable to mount an adequate response to thermal stress. Total proteome analysis of wild-type and AlaRS C666A strains further revealed that in the absence of AlaRS proofreading, more than 800 proteins were differentially enriched or under-represented [13]. Despite the observation that serine mistranslation was still occurring in the suppressor mutants, it was of interest to determine if the phenotypes associated with the proteome dysregulation in the AlaRS C666A strain were still present in the suppressor strains. To determine if the suppressor strains were able to respond to a shift in thermal stress, the suppressor strains were grown in LB media at 42 \({}^{\circ}\)C. As had been previously observed [13], the AlaRS C666A strain has an elevated growth sensitivity at 42 \({}^{\circ}\)C compared to 37 \({}^{\circ}\)C (Figures 2A and 4A). However, none of the single or double suppressor mutants elicited any deviation in growth when compared to wild-type _E. coli_ (Figure 4A).