Specificity in the biosynthesis of the universal tRNA nucleoside N(6)-threonylcarbamoyl adenosine (t(6)A)—TsaD is the gatekeeper

N(6)-threonylcarbamoyl adenosine (t(6)A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t(6)A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO(2), and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A(37) of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn(6)A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t(6)A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t(6)A pathway, we carried out biochemical and biophysical investigations of the t(6)A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn(6)A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn(6)A in their tRNA, we conclude that their origin is due to formation via the t(6)A pathway.