Modification of synthetic RNA substrates by TkNat10

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification conserved across all domains of life. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. This dataset uses a high-throughput mutagenesis approach to define sequence and structural determinants necessary for TkNat10-catalyzed modification of model SSU rRNA substrates. These findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.