The RNA acetyltransferase driven by ATP hydrolysis synthesizes N ⁴-acetylcytidine of tRNA anticodon

The wobble base of Escherichia coli elongator tRNA^Met is modified to N⁴-acetylcytidine (ac⁴C), which is thought to ensure the precise recognition of the AUG codon by preventing misreading of near-cognate AUA codon. By employing genome-wide screen of uncharacterized genes in Escherichia coli ('ribonucleome analysis'), we found the ypfI gene, which we named tmcA (tRNA^Met cytidine acetyltransferase), to be responsible for ac⁴C formation. TmcA is an enzyme that contains a Walker-type ATPase domain in its N-terminal region and an N-acetyltransferase domain in its C-terminal region. Recombinant TmcA specifically acetylated the wobble base of E. coli elongator tRNA^Met by utilizing acetyl-coenzyme A (CoA) and ATP (or GTP). ATP/GTP hydrolysis by TmcA is stimulated in the presence of acetyl-CoA and tRNA^Met. A mutation study revealed that E. coli TmcA strictly discriminates elongator tRNA^Met from the structurally similar tRNA^Ile by mainly recognizing the C27-G43 pair in the anticodon stem. Our findings reveal an elaborate mechanism embedded in tRNA ^Met and tRNA^Ile for the accurate decoding of AUA/AUG codons on the basis of the recognition of wobble bases by the respective RNA-modifying enzymes.