## 3 Results
### In Vitro Characterization of the AlaRS C666A Suppressors  
Having previously demonstrated that the AlaRS C666A suppressors do not prevent mistranslation in vivo, biochemical analyses were performed to potentially identify differences in the formation of the Ser-tRNA\({}^{\text{Ala}}\) substrates in vitro. Recombinant AlaRS variants were expressed and purified from _E. coli_ and in vitro transcribed _E. coli_ tRNA\({}^{\text{Ala}}\) was used as a tRNA substrate for all cognate and non-cognate experimentation. To determine if there were any differences in non-cognate Ser-tRNA  
Figure 4: Suppressor mutants alleviate AlaRS C666A-associated phenotypes. Second-site suppressors alleviated the (**A**) heat stress, (**B**) swimming, and (**C**) antibiotic sensitivities previously observed in the AlaRS C666A mutant. Statistical significance was determined by one-way ANOVA with Tukey post-hoc comparison (*_p_ \(<\) 0.0005). formation, in vitro mis-aminoacylation experiments were performed using all of the AlaRS variants. The AlaRS C666A editing-defective variant was able to mis-servlate tRNA\({}^{\text{Ala}}\) as expected, and all of the AlaRS suppressor enzymes were also able to mis-aminoacylate tRNA\({}^{\text{Ala}}\) (Figure 5A). While the AlaRS suppressor variants were able to mis-servlate tRNA\({}^{\text{Ala}}\), AlaRS C666A R561C did exhibit a \(\sim\)50% reduction in Ser-tRNA\({}^{\text{Ala}}\) product formation (Figure 5B). Consistent with observations using the in vivo mistranslation reporter, this in vitro analysis suggests that the AlaRS suppressor variants are unable to prevent mis-aminoacylation but a substitution of R561C can potentially lead to an overall decrease in mis-servlated tRNA formation in the cell. Figure 5: In vitro characterization of AlaRS C666A suppressors. The three identified AlaRS C666A suppressors were assayed for (**A**) mis-servation activity and results from the (**B**) AlaRS R561C mutants were further analyzed. The proofreading activity of the enzymes were examined over a time course for (**C**) Ser-tRNA\({}^{\text{Ala}}\) and (**D**) Ala-tRNA\({}^{\text{Ala}}\) substrates. Furthermore the initial rates of Ser-tRNA\({}^{\text{Ala}}\) deactivation were monitored for the (**E**) single mutants and (**F**) double mutants. Finally, ATP consumption was observed for reactions including tRNA\({}^{\text{Ala}}\) and (**G**) serine or (**H**) alanine. In addition to the direct contribution of Ser-tRNA\({}^{\text{Ala}}\) product formation, it was possible that the mechanism for the suppressors could be occurring due to changes in Ser-tRNA\({}^{\text{Ala}}\) proofreading. Pre-formed Ser-tRNA\({}^{\text{Ala}}\) was incubated with all of the AlaRS variants and deactivation was monitored over time. None of the second-site suppressors were able to proofread the mis-servylated tRNA product similar to wild-type AlaRS levels (Figure 5C). The AlaRS R561S variant enzyme exhibited elevated levels of cognate Ala-tRNA\({}^{\text{Ala}}\) deacylation, with the percent of deacylated substrates reaching \(\sim\)40% after 30 min (Figure 5D). Upon further inspection of the initial rates of Ser-tRNA\({}^{\text{Ala}}\) deacylation, two of the single mutant suppressors, AlaRS D551Y and AlaRS R561S appear to be proofreading faster than the wild-type enzyme (Figure 5E). Furthermore, evaluation of the initial deacylation rates for the double mutants suggests that AlaRS D551Y C666A and the AlaRS R561S C666A enzymes have slightly elevated proofreading activities compared to the editing-defective AlaRS C666A enzyme (Figure 5F). An additional observation made from the in vitro deacylation experiment is that the single AlaRS R561C variant was unable to deacylate Ser-tRNA\({}^{\text{Ala}}\) in trans.