Structure and mechanism of a bacterial t6A biosynthesis system

Abstract

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 structure of the TsaB/TsaD/TsaE complex reveals an unexpected mechanism for the bacterial t6A tRNA-modification

“…Sequence alignments with superposed secondary structure assignments of a few TsaB, TsaD and TsaE orthologues are represented in Supplementary Figure S1 . As illustrated in Figure 2a , Tm TsaB and Tm TsaD embrace Tm TsaE to form a compact ternary complex, in agreement with SAXS measurements on Ec - and Tm TsaBDE in solution ( 22 , 23 ). Tm TsaE is held in a grip between the helical insertion of the C-terminal domain of Tm TsaD on one side and Tm TsaB on the other.…”

“…In a very recent manuscript, Luthra et al . reported the characterization of the TsaBDE threonylcarbamoyl transfer complex of T. maritima ( Tm TsaBDE ) ( 23 ). Their SAXS analysis revealed that Tm TsaBDE forms a symmetric hexameric quaternary assembly in solution with 2:2:2 stoichiometry ( Tm TsaB 2 D 2 E 2 ).…”

“…Tm TasB and Tm TsaD were further shown to form a Tm TsaB 2 D 2 complex that was capable to bind one tRNA molecule, and accommodate a single TsaE subunit. Quantitative activity measurements demonstrated that Tm TsaB 2 D 2 alone can catalyse only a single round of t 6 A synthesis ( 23 ). An ensemble of kinetic and binding experiments led to the hypothesis that Tm TsaE-catalyzed ATP hydrolysis occurs after the release of the t 6 A-modifed tRNA ( 23 ).…”

“…The AMPCPP nucleotide was found sandwiched between Tm TsaE and Tm TsaD with its γ-phosphate group being coordinated by side chains from both subunits. Crystallographic symmetry generates the hexameric Tm TsaB 2 D 2 E 2 complex that was proposed to be present in solution ( 23 ). Surprisingly, Tm TsaE partially denatures the active site of Tm TsaD and prevents simultaneous substrate tRNA binding, suggesting that Tm TsaE is required for a reaction step before or after the TC-transfer from TC-AMP to tRNA.…”

<i>In Silico</i> Structural and Functional Annotation of Nine Essential Hypothetical Proteins from <i>Streptococcus pneumoniae</i>

“…This protein is indirectly responsible in the N6threonylcarbamoyladenosine (t(6)A) pathway. A study found that t6A involves in decoding accuracy of mRNA codons during protein synthesis [42]. Evidently, a defect in t(6)A pathway can lead to increased frame shift events, wrong start codon selections and occurrence of pleiotropic phenotypes [43].…”