## 3 Results  
### Identification of Suppressor Mutations That Alleviate the AlaRS C666A Growth Defect  
It has been previously observed that perturbation of AlaRS proofreading in _E. coli_ leads to growth defects when compared to wild-type _E. coli_[13]. To determine if secondary mutations in the _E. coli_ genome could suppress the growth defects associated with a deficiency in AlaRS proofreading, suppressor mutants that restore near-normal growth compared to their proofreading-deficient strain were screened for. Due to the severe growth defects associated with AlaRS-mediated mistranslation, suppressor mutations were anticipated to act in reducing the mistranslational load on the proteome. It was speculated that this suppression could possibly occur by increasing the thermodynamic barrier for EF-Tu discrimination and thus preventing mis-acylated tRNA accumulation into an active ternary complex [22; 23; 24]. Additionally, the _E. coli_ protein ProXP-ST1 was previously shown to have activity against Ser-tRNA\({}^{\text{Ala}}\) in vitro [25]. Therefore it was possible that mutations in the _yeaK_ gene (gene encoding ProXP-ST1) could increase ProXP-ST1 proofreading activity on mis-servylated tRNA\({}^{\text{Ala}}\) substrates. Suppressor colonies of the MG1655 AlaRS C666A strain accumulated on LB agar plates without the need for additional mutagenic manipulation. One initial wild-type-like colony was selected from the mixed population, colony purified, and genomic DNA (gDNA) was extracted. The isolated gDNA was further subjected to whole-genome sequencing (WGS) for variant analysis. When compared to another control sample that was also analyzed by WGS, only one additional mutation was identified in the suppressor mutant. The identified single nucleotide polymorphism (SNP) was a G1651T mutation in the _alaS_ gene that when translated, caused a D551Y substitution in AlaRS. Having identified second-site _alaS_ suppressors, two additional suppressor mutant colonies were isolated from LB agar plates. Rather than subjecting the two additional mutants to WGS analysis, gDNA was extracted from these mutants and the DNA was used as the template for PCR to amplify the _alaS_ gene. This _alaS_ amplicon was used as the template for a Sanger sequencing reaction using a primer with homology to _alaS_. Sanger sequencing of the two additional suppressors reported that both of the mutants also contained secondary mutations in the _alaS_ gene, with both SNPs occurring at nucleotide position C1681, ultimately resulting in two different substitutions at R561, R561C, and R561S. While the _alaS_ gene encodes one complete monomeric unit of AlaRS, this gene contains at least three characterized domains responsible for distinct enzymatic activity including, amino acid activation and tRNA aminoacylation [26; 27], proofreading [9], and a C-terminal C-Ala domain required for oligomerization that also participates in aminoacylation [28; 29]. The three suppressor mutations all occur in the proofreading domain of AlaRS (Figure 1A). To date, these two amino acid positions have yet to be characterized or predicted to participate in any proofreading activity. The coordination of the proofreading domain active site has previously been explored and has indicated that residues involved in mis-acylated tRNA hydrolysis are evolutionarily conserved (e.g., C666 in _E. coli_) [30]. To determine if the two amino acid residues identified in the suppressor screen are also conserved, AlaRS proofreading domains were aligned across several prokaryotic and eukaryotic model systems. With the exception of the closely related _Salmonella enterica_, these amino acid positions do not appear to be evolutionarily conserved (Figure 1B). Currently, no structural data for the _E. coli_ AlaRS proofreading domain exists. Using Phyre2 modeling predictions, the position of the suppressor residues were mapped on the AlaRS structure.