Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3

The essential deamination of adenosine A(34) to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A(34) deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome. The deamination of all adenosines in the wobble base position is essential to empower the individual tRNA to decode several codons. Here, the authors present the cryo-EM structure of the tRNA-bound ADAT2/3 deaminase, revealing how the geometry-specific enzyme acquiesces sequence-divergent tRNAs to its active site.