The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t6A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteria.

The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

The universal N(6)‐threonylcarbamoyladenosine (t6A) modification at position 37 of ANN‐decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD, and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here we report biochemical and biophysical studies of the t6A biosythesis system from Thermotoga maritima. Small angle X‐ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB2D2 complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB2D2 alone, and a TsaB2D2E1 complex with TsaE mutants deficient in ATPase activity, can catalyze only a single cycle of t6A synthesis, providing evidence that TsaE‐catalyzed ATP hydrolysis is responsible for the turnover of product tRNA. Based on these results, we propose a model for t6A biosynthesis in bacteriaSupport or Funding InformationThis work was supported by the National Institutes of Health (GM11058)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Archaeosine (G+) is a highly modified nucleosides found exclusively at position‐15 in the dihydrouridine loops (D‐loop) of archaeal tRNAs where it contributes to tRNA structural stability. In a late step in G+ biosynthesis, the precursor nucleoside 7‐cyano‐7‐deazaguanine (preQ0) is inserted into tRNA by the enzyme tRNA‐guanine transglycosylase (arcTGT) before conversion to G+ by archaeosine synthases that vary among archaeal phyla: ArcS in Euryarchaeota, and QueF‐like or GAT‐QueC in Chrenoarchaeota. GAT‐QueC is a two‐domain enzyme harboring an N‐terminal glutamine amidotransferase (GAT) domain, and a C‐terminal domain homologous to QueC, the enzyme that produces preQ0. Consistent with its 2‐domain structure, GAT‐QueC functions in vivo as both a preQ0 synthase and an amidinotransferase, generating G+‐modified tRNA. However, it remains unknown whether the GAT domain acts on preQ0 before or after its insertion into tRNA by arcTGT. Here we describe overexpression, purification and preliminary biochemical and crystallographic characterization of GAT‐QueC from Sulfolobus tokodaii (StGAT‐QueC). Analytical gel filtration chromatography and electrophoretic mobility shift analysis reveal that StGAT‐QueC is a monomeric protein with molecular weight of 55 kDa and no affinity for tRNA, suggesting that the enzyme acts on free preQ0, not preQ0‐modified tRNA. High throughput crystallization of the apoenzyme (and in the presence of preQ0) using the vapor diffusion method yielded crystals that grew in 21 days from solutions containing methyl pentanediol and sodium acetate, and diffracted to 5.8 Å without cryo protection. The crystals belong to cubic space group F23, exhibit unit cell length=292 Å and contain 4 protein molecules per asymmetric unit. Support or Funding Information NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).

Two important modifications of tRNA, the 7‐deazaguanine nucleosides queuosine (Q) and archaeosine (G+), are biosynthesized from GTP in bacteria and archaea, respectively, in a well characterized multi‐enzyme pathway leading to the shared advanced intermediate, 7‐cyano‐7‐deazaguanine (preQ0). In bacteria, preQ0 is converted to an aminomethyl derivative that is then inserted in tRNA by the bacterial tRNA‐guanine transglycosylase (TGT) enzyme. In archaea, preQ0 is inserted directly in tRNA by the archaeal TGT before conversion to G+. Recently, in 230 bacterial and phage genomes, a genomic island that contains a paralog of TGT was identified, which led to the discovery of Q, G+ and preQ0 deoxy derivatives in the DNA of some of these organisms. This gene cluster was renamed DpdA‐K, for “7‐deazapurine in DNA.” The G+‐modified DNA of E. coli bacteriophage 9g has been shown to resist restriction by >140 Type II restriction endonucleases (REases), consistent with a role of the modification as a defense mechanism. Recent experiments have shown that DpdA is a DNA‐guanine transglycosylase that catalyzes the insertion of preQ0 at a specific palindromic sequence in DNA. Here we report overexpression, purification, and crystallographic analysis of S. Montevideo DpdA (SmDpdA, ~47 kDa). The crystal structure, determined at 2.25‐Å resolution by multi‐wavelength anomalous diffraction methods, reveals a TIM‐barrel enzyme similar to the bacterial TGT in overall fold, active site and structural Zn2+ site. However, a large DpdA‐specific insertion in the TIM barrel provides an extended positively charged 26‐Å wide surface groove, consistent with a DNA binding surface. Free docking of a DNA duplex using the HADDOCK server places DNA to this surface in a fashion reminiscent of DNA binding to the ubiquitous Zn2+‐dependent DNA repair enzyme apurinic/apyrimidinic endonuclease IV. The structure and docking model suggest that DpdA bends its substrate DNA by 60° and flips the modification‐site guanine base out of the helix for transglycosylation. The results also inform the design of a substrate DNA duplex for future crystallization of the nucleoprotein complex. Support or Funding Information NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).