Stoichiometry of triple-sieve tRNA editing complex ensures fidelity of aminoacyl-tRNA formation

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids onto tRNAs. To avoid mistranslation, editing mechanisms evolved to maintain tRNA aminoacylation fidelity. For instance, while rejecting the majority of non-cognate amino acids via discrimination in the synthetic active site, prolyl-tRNA synthetase (ProRS) misactivates and mischarges Ala and Cys, which are similar in size to cognate Pro. Ala-tRNA(Pro) is specifically hydrolyzed by the editing domain of ProRS in cis, while YbaK, a free-standing editing domain, clears Cys-tRNA(Pro) in trans. ProXp-ala is another editing domain that clears Ala-tRNA(Pro) in trans. YbaK does not appear to possess tRNA specificity, readily deacylating Cys-tRNA(Cys)in vitro. We hypothesize that YbaK binds to ProRS to gain specificity for Cys-tRNA(Pro) and avoid deacylation of Cys-tRNA(Cys) in the cell. Here, in vivo evidence for ProRS-YbaK interaction was obtained using a split-green fluorescent protein assay. Analytical ultracentrifugation and native mass spectrometry were used to investigate binary and ternary complex formation between ProRS, YbaK, and tRNA(Pro). Our combined results support the hypothesis that the specificity of YbaK toward Cys-tRNA(Pro) is determined by the formation of a three-component complex with ProRS and tRNA(Pro) and establish the stoichiometry of a ‘triple-sieve’ editing complex for the first time.