## 1 Introduction  
Across all domains of life, protein-coding sequences have evolved to maintain activity of essential biological processes. Specific residues or modular domains with high evolutionary conservation within a polypeptide sequence can provide insight into regions of functional essentiality of a given protein [1, 2]. Because of this, mechanisms have also evolved to minimize errors in protein synthesis [3].  
Translational fidelity is maintained at several distinct stages of protein synthesis, with one of the key steps being the accuracy of aminoacyl-tRNA synthesis by aaRSs. AaRSs are responsible for pairing free amino acids in the cell to their cognate tRNAs, with the product then released for participation in ternary complex formation with elongation factor and GTP [4]. This complex is then recruited to the A-site of the ribosome to facilitate peptide bond formation. Most organisms encode at least one distinct aaRS gene for each of the twenty proteinogenic amino acids in the cell. In complex with ATP, aaRSs bind to free cognate amino acids leading to the synthesis of an aminoacyl-adenylate. This activated amino acid will then be transferred to the 3' end of the tRNA to be released in the cell for translation. AaRSs have evolved mechanisms to prevent the accumulation of mis-aminoacylated tRNAs. Half of the aaRSs in _Escherichia coli_ are able to occlude non-cognate amino acid activation in the primary active site; however, the other ten aaRS will mis-activate non-cognate substrates [5]. To prevent non-cognate aa-tRNAs from being released into the cell, many aaRSs have evolved secondary mechanisms to hydrolyze non-cognate products. Several synthetases, including isoleucyl-tRNA synthetase have evolved hydrolytic activity against non-cognate amino acids at the amino acid activation site. Upon non-cognate adenylate recognition, the amino acid and AMP will be hydrolyzed and released back into the cell [6]. In contrast, several aaRS including, phenylalanine-tRNA synthetase (PheRS) [7], threnyl-tRNA synthetase (ThrRS) [8], and alanyl-tRNA synthetase (AlaRS) [9] encode a distinct secondary active site which contains proofreading activity against non-cognate amino acid-containing substrates after the amino acid is transferred to the 3' end of the tRNA.  
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\({}^{\text{Ala}}\) 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. In this work, we identify and characterize two additional residues in _E. coli_ AlaRS that play roles in enzymatic activity. All of the novel suppressors appear to modulate AlaRS fidelity through unique and distinct mechanisms. Furthermore, the observations presented herein suggest that a slight increase in AlaRS proofreading activity may be sufficient to alleviate the AlaRS C666A-associated physiological defects. More broadly, this work demonstrates the utility of applying genetic approaches to further elucidate novel mechanistic features of aaRSs.