Discovery and Characterization of the Proteins Involved in the Synthesis of N⁶-Threonylcarbamoyl Adenosine, a Nucleoside Modification of tRNA

N6-threonylcarbamoyl adenosine (t6A) is a universally conserved tRNA modification found at position 37 of tRNAs which decode ANN codons. Structural studies have implicated its presence as a requirement for the disruption of a U-turn motif in certain tRNAs, leading to the formation of properly structured anticodon stem loop. This structure is proposed to enhance the base pairing between U36 of tRNA and A1 of the codon which aids in translational frame maintenance. Despite significant effort since its discovery in the 1970s the enzymes involved in its biosynthesis remained undiscovered. Bioinformatic analysis identified two proteins as likely candidates for t6A synthesis, YrdC and YgjD. Subsequent gene knockout experiments in yeast were consistent with their involvement in t6A biosynthesis in vivo. Furthermore, clustering between the bacterial genes ygjD, yeaZ and yjeE as well as the identification of a protein interaction network between YgjD, YeaZ, and YjeE suggested that YeaZ and YjeE might be involved in t6A biosynthesis. The genes encoding ygjD, yeaZ, yrdC and yjeE were cloned from E. coli and the recombinant protein was purified. Experiments analyzing the incorporation of [U-14C]-L-threonine and [14C]-bicarbonate (substrates previously indicated in its biosynthesis) into tRNA in the presence of these four proteins demonstrated the first reconstitution of the t6A pathway in vitro. LC-MS analysis verified the formation of t6A, and these proteins were renamed TsaD (YgjD), TsaB (YeaZ), TsaC (YrdC), and TsaE (YjeE). Biochemical characterization of this pathway suggested that the formation of t6A proceeds through an unstable threonylcarbamoyl adenosine monophosphate (TC-AMP) intermediate, which is produced by TsaC from its substrates CO2, L-threonine and ATP. To investigate this reaction in more detail a coupled assay was developed, enabling sensitive detection of turn over. TsaC is a promiscuous enzyme which readily accepts several amino acids as substrates. The formation of t6A from TC-AMP is catalyzed by TsaD, TsaB, and TsaE. Of these three proteins only TsaD is universally conserved suggesting it is the protein catalyzing the transfer of the threonylcarbamoyl moiety to A37 of tRNA. This transfer is not promiscuous as only TC-AMP serves as an efficient substrate for t6A formation. Structural investigation of these proteins are consistent with the formation of a single protein complex potentially alleviating issues with the reactivity and instability of TC-AMP