Mammalian trans-editing factor ProX is able to deacylate tRNAThr mischarged with alanine.

The precise coupling of tRNAs with their cognate amino acids, known as tRNA aminoacylation, is a stringently regulated process that governs translation fidelity. To ensure fidelity, organisms deploy multiple layers of editing mechanisms to correct mischarged tRNAs. Prior investigations have unveiled the propensity of eukaryotic AlaRS to erroneously attach alanine onto tRNACys and tRNAThr featuring the G4:U69 base pair. In light of this, and given ProXp-ala's capacity in deacylating Ala-tRNAPro, we embarked on exploring whether this trans-editing factor could extend its corrective function to encompass these mischarged tRNAs. Our in vitro deacylation assays demonstrate that murine ProXp-ala (mProXp-ala) is able to efficiently hydrolyze Ala-tRNAThr, while Ala-tRNACys remains unaffected. Subsequently, we determined the first structure of eukaryotic ProXp-ala, revealing a dynamic helix α2 involved in substrate binding. By integrating molecular dynamics simulations and biochemical assays, we pinpointed the pivotal interactions between mProXp-ala and Ala-tRNA, wherein the basic regions of mProXp-ala as well as the C3-G70 plays essential role in recognition. These observations collectively provide a cogent rationale for mProXp-ala's deacylation proficiency against Ala-tRNAThr. Our findings offer valuable insights into the translation quality control within higher eukaryotic organisms, where the fidelity of translation is safeguarded by the multi-functionality of extensively documented proteins.