tRNA, the central molecule of translation, is heavily modified post transcriptionally with noncanonical nucleosides that constitute ~83% of all RNA modifications. Modifications of the anticodon stem‐loop (ASL) of tRNA are required for translational fidelity and codon diversification. The ASL modification N6‐threonylcarbamoyl adenosine (t6A) at position 37 in ANN‐decoding tRNAs (N is any nucleobase) is universally conserved and its deficiency in humans causes renal‐neurological disease. Due to its essentiality in bacteria, the t6A pathway has emerged as a potential antibacterial target. Bacterial t6A biosynthesis begins with synthesis of the intermediate threonylcarbamoyl adenylate (TC‐AMP) from threonine, ATP and bicarbonate by the TsaC enzyme, followed by transfer of the threonylcarbamoyl moiety from TC‐AMP to A37 of substrate tRNA by the theonylcarbamoyl transfer (TCT) complex, comprised of the three proteins TsaB, TsaD and TsaE. We recently showed in T. maritima that while the TsaBD complex is the substrate binding platform responsible for TC transfer, the complex becomes inactivated after each t6A synthesis cycle, and TsaE‐dependent ATP hydrolysis “resets” the platform for multi‐turnover catalysis. However, the nature of the inactivation and reset steps remain unclear. Recent reports of a kinase activity for TsaE prompted us to investigate the role of this activity in the t6A cycle. Using a radiochemical kinase assay and mutagenesis experiments we show that T. maritima TsaE switches between a highly stable multimeric form possessing a tyrosine autokinase activity and a monomeric form lacking kinase activity and functioning as an ATPase in t6A synthesis. Further, to elucidate the structural basis of the specificity of t6A biosynthesis to ANN‐decoding tRNA substrates, we initiated cryo‐EM studies of the tRNA‐bound TCT complex. The preliminary maps reveal tRNA bound on the back surface of the TsaD subunit with its anticodon lodged in the TC‐transfer site and acceptor stem stabilized by the C‐terminal tail of TsaB, consistent with previous biochemical data. Collectively, the results provide insight into the mechanism of TC transfer, and suggest a regulatory role of TsaE that links t6A synthesis to other cellular processes.Support or Funding InformationFunding: NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).