The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis

Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 angstrom resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.

Automated summary

Thiolation of uridine 34 in tRNAs is conserved in all three domains of life and is essential for accurate protein translation. The mechanism of U34-tRNA thiolation involves a [4Fe-4S] cluster, which is coordinated by conserved cysteine residues. The enzyme NcsA in archaea and the Ctu1/Ctu2 complex in eukaryotes play a crucial role in catalyzing U34-tRNA thiolation. The crystal structure of NcsA from Methanococcus maripaludis reveals the coordination of the [4Fe-4S] cluster and provides insights into the binding and activation of the sulfur donor.