The Elongator subunit Elp3 is a non-canonical tRNA acetyltransferase

The Elongator complex catalyzes posttranscriptional tRNA modifications by attaching carboxy-methyl (cm 5 ) moieties to uridine bases located in the wobble position. The catalytic subunit Elp3 is highly conserved and harbors two individual subdomains, a radical S -adenosyl methionine (rSAM) and a lysine acetyltransferase (KAT) domain. The details of its modification reaction cycle and particularly the substrate specificity of its KAT domain remain elusive. Here, we present the co-crystal structure of bacterial Elp3 (DmcElp3) bound to an acetyl-CoA analog and compare it to the structure of a monomeric archaeal Elp3 from Methanocaldococcus infernus (MinElp3). Furthermore, we identify crucial active site residues, confirm the importance of the extended N-terminus for substrate recognition and uncover the specific induction of acetyl-CoA hydrolysis by different tRNA species. In summary, our results establish the clinically relevant Elongator subunit as a non-canonical acetyltransferase and genuine tRNA modification enzyme. Elp3 is the catalytic subunit of the eukaryotic Elongator complex that catalyzes posttranscriptional tRNA modifications. Here the authors present the crystal structures of an acetyl-CoA analog bound bacterial Elp3 and a monomeric archaeal Elp3 and show that Elp3 functions as a tRNA modification enzyme in all domains of life.