The C-Terminal Domain of the Archaeal Leucyl-tRNA Synthetase Prevents Misediting of Isoleucyl-tRNAIle

Abstract

In the archaeal leucyl-tRNA synthetase (LeuRS), the C-terminal domain recognizes the long variable arm of tRNA^Leu for aminoacylation, and the so-called editing domain deacylates incorrectly formed Ile-tRNA^Leu. We previously reported, for Pyrococcus horikoshii LeuRS, that a deletion mutant lacking the C-terminal domain (LeuRS_Δ(811−967)) retains normal editing activity, but has severely reduced aminoacylation activity. In this study, we found that LeuRS_Δ(811−967), but not the wild-type LeuRS, exhibited surprisingly robust deacylation activity against Ile-tRNA^Ile, correctly formed by isoleucyl-tRNA synthetase (“misediting”). Structural superposition of tRNA^Ile onto the LeuRS·tRNA^Leu complex indicated that Ile911, Lys912, and Glu913 of the LeuRS C-terminal domain clash with U20 of tRNA^Ile, which is bulged out as compared to the corresponding nucleotide of tRNA^Leu. The deletion of amino acid residues 911−913 of LeuRS enhanced the Ile-tRNA^Ile deacylation activity, without affecting the Ile-tRNA^Leu deacylation activity. These results demonstrate that the clashing between U20 of tRNA^Ile and residues 911−913 of the LeuRS C-terminal domain is the structural mechanism that prevents misediting. In contrast, the deletion of the C-terminal domains of the isoleucyl- and valyl-tRNA synthetases impaired both the aminoacylation (Ile-tRNA^Ile and Val-tRNA^Val formation, respectively) and editing (Val-tRNA^Ile and Thr-tRNA^Val deacylation, respectively) activities, and did not cause misediting (Val-tRNA^Val and Thr-tRNA^Thr deacylation, respectively) activity. Thus, the requirement of the C-terminal domain for misediting prevention is unique to LeuRS, which does not recognize the anticodon of the cognate tRNA, unlike the common aminoacyl-tRNA synthetases.