Recent Advances in Our Understanding of the Biosynthesis of Sulfur Modifications in tRNAs

Sulfur is an essential element in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, introduced post-transcriptionally, that function to ensure proper codon recognition or stabilization of tRNA structure, thereby enabling accurate and efficient translation. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems that are closely related to cellular sulfur metabolism, and “modification enzymes” that incorporate sulfur atoms into tRNA. Herein, recent biochemical and structural characterization of the biosynthesis of sulfur modifications in tRNA is reviewed, with special emphasis on the reaction mechanisms of modification enzymes. It was recently revealed that TtuA/Ncs6-type 2-thiouridylases from thermophilic bacteria/archaea/eukaryotes are oxygen-sensitive iron-sulfur proteins that utilize a quite different mechanism from other 2-thiouridylase subtypes lacking iron-sulfur clusters such as bacterial MnmA. The various reaction mechanisms of RNA sulfurtransferases are also discussed, including tRNA methylthiotransferase MiaB (a radical S-adenosylmethionine-type iron-sulfur enzyme) and other sulfurtransferases involved in both primary and secondary sulfur-containing metabolites

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s(2)T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed. Chen et al. demonstrate how the Fe-S cluster receives sulfur from TtuB, a ubiquitin-like sulfur donor during tRNA modification. They find that the release of sulfur from the thiocarboxylated C-terminus of TtuB depends on the adenylation of the substrate tRNA. This study provides molecular insights into the sulfur modification of tRNA

Common thiolation mechanism in the biosynthesis of tRNA thiouridine and sulphur-containing cofactors

2-Thioribothymidine (s2T), a modified uridine, is found at position 54 in transfer RNAs (tRNAs) from several thermophiles; s2T stabilizes the L-shaped structure of tRNA and is essential for growth at higher temperatures. Here, we identified an ATPase (tRNA-two-thiouridine C, TtuC) required for the 2-thiolation of s2T in Thermus thermophilus and examined in vitro s2T formation by TtuC and previously identified s2T-biosynthetic proteins (TtuA, TtuB, and cysteine desulphurases). The C-terminal glycine of TtuB is first activated as an acyl-adenylate by TtuC and then thiocarboxylated by cysteine desulphurases. The sulphur atom of thiocarboxylated TtuB is transferred to tRNA by TtuA. In a ttuC mutant of T. thermophilus, not only s2T, but also molybdenum cofactor and thiamin were not synthesized, suggesting that TtuC is shared among these biosynthetic pathways. Furthermore, we found that a TtuB–TtuC thioester was formed in vitro, which was similar to the ubiquitin-E1 thioester, a key intermediate in the ubiquitin system. The results are discussed in relation to the mechanism and evolution of the eukaryotic ubiquitin system