Specificity in the biosynthesis of the universal tRNA nucleoside N⁶-threonylcarbamoyl adenosine (t⁶A)—TsaD is the gatekeeper

Abstract

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.

Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli

“…The substrate inhibition observed is consistent with a similar observation recently reported by Swinehart et al. ( 58 ). Kinetic data with tRNA as the variable substrate was somewhat limited by the apparent low values of K m for most tRNA transcripts.…”

“…In vitro substrate activity of hNV with SUA5 and KEOPS has been shown in the archaeal system by Perrochia, et al ( 31 ) and also recently shown for E. coli and T. maritima by Swinehart et al. ( 58 ). In the structure, the binding pocket for the threonine side chain has an open face into the ‘tunnel’ formed in TsaD such that extended alkyl chains may be accommodated if they can project into the tunnel but not if they are restricted in the direction of Cys10.…”

“…The Ser9Gly, Cys10Gly double mutant is inactive, which indicates additive effects of removing van der Waals interactions with the threonine moiety. These mutants suggest that the specificity for the l -threonyl part of TC-AMP is enforced by a number of weak interactions that together lead to selection of threonine/hydroxynorvaline over other potential amino acids and provides a structural explanation for the recent report of TsaD as the main guardian of such specificity ( 58 ).…”

Modified base-binding EVE and DCD Domains Implicated in the Origins of Programmed Cell Death and the piRNA Pathway

“…The most prominent contextual association of the EVE domain observed in our study is its inclusion in the putative operon TsaD->GpsA->YciI->EVE in α-proteobacteria. TsaD modifies tRNAs that decode ANN codons (Met, Ile, Thr, Asn, Lys, Arg, Ser) to introduce threonylcarbamoyladenosine (t 6 A) at position 37, immediately adjacent to the anticodon (Swinehart et al 2020). t 6 A is a universal modification that is essential for translational fidelity, and mutations in this pathway lead to errors in start codon selection and aberrant frameshifts (Missoury et al 2018;Swinehart et al 2020;Deutsch et al 2012).…”

“…TsaD modifies tRNAs that decode ANN codons (Met, Ile, Thr, Asn, Lys, Arg, Ser) to introduce threonylcarbamoyladenosine (t 6 A) at position 37, immediately adjacent to the anticodon (Swinehart et al 2020). t 6 A is a universal modification that is essential for translational fidelity, and mutations in this pathway lead to errors in start codon selection and aberrant frameshifts (Missoury et al 2018;Swinehart et al 2020;Deutsch et al 2012). The α-proteobacterial glycerol-3-phosphate dehydrogenase that is tightly associated with EVE is orthologous to the corresponding mitochondrial enzyme that contributes electrons to the respiratory chain (Mráček, Drahota, and Houštěk 2013).…”