Gly56 in the synthetic site of isoleucyl-tRNA-synthetase confers specificity and maintains communication with the editing site.

Isoleucyl-tRNA synthetase (IleRS) links isoleucine to cognate tRNA via the Ile-AMP intermediate. Non-cognate valine is often mistakenly recognized as the IleRS substrate; therefore, to maintain the accuracy of translation, IleRS hydrolyzes Val-AMP within the synthetic site (pre-transfer editing). As this activity is not efficient enough, Val-tRNA Ile is formed and hydrolyzed in the distant post-transfer editing site. A strictly conserved synthetic site residue Gly56 was previously shown to safeguard Ile-to-Val discrimination during aminoacyl (aa)-AMP formation. Here, we show that the Gly56Ala variant lost its specificity in pre-transfer editing, confirming that this residue ensures the selectivity of all synthetic site reactions. Moreover, we found that the Gly56Ala mutation affects IleRS interaction with aa-tRNA likely by disturbing tRNA-dependent communication between the two active sites.