Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a protein-bound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.

Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron–sulfur [Fe–S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe–4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s²U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe–S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.

In most organisms, the widely conserved 1-methyl-adenosine58 (m¹A₅₈) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m¹A₅₇ being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (_PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. _PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A₅₇ but not A₅₈, confirming that _PabTrmI methylates efficiently the first adenine of the A₅₇A₅₈A₅₉ sequence. _PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m¹A₅₈ formation triggers RNA release. A model of the protein–tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.