Elp3 uses a conserved molecular tunnel to transport acetate between distant active sites and catalyze tRNA wobble...

The radical SAM enzyme Elp3 and eukaryotic Elongator complex catalyze formation of a key intermediate transfer RNA (tRNA) modification, 5-carboxymethyluridine (cm⁵U), in the anticodons of tRNAs across all domains of life. cm⁵U-derived modifications are important for fine tuning codon-anticodon interactions and efficient protein translation, and defects in this modification are linked to development of neurodegenerative disease in humans. Here we reconstitute tRNA modification activity with a model Elp3 enzyme and combine structural analyses, enzymology, and isotope incorporation experiments to show Elp3 harbors a conserved molecular tunnel that shuttles free acetate molecules from the acetyl-CoA binding domain to the radical SAM active site over 20 Å away, where acetate undergoes radical-mediated reaction and addition to tRNA U34. Our model explains how Elp3 bridges a large distance between active sites to catalyze tRNA carboxymethylation and illustrates a unique mechanism for intermediate transport in radical SAM enzymes.