Sulfur is an essential element for a variety of cellular constituents in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2‑thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A). Earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mRNA or stabilization of tRNA structure. In many cases, the biosynthesis of these sulfur modifications starts with cysteine desulfurases, which catalyze the generation of persulfide (an activated form of sulfur) from cysteine. Many sulfur-carrier proteins are responsible for delivering this activated sulfur to each biosynthesis pathway. Finally, specific “modification enzymes” activate target tRNAs and then incorporate sulfur atoms. Intriguingly, the biosynthesis of 2-thiouridine in all domains of life is functionally and evolutionarily related to the ubiquitin-like post-translational modification system of cellular proteins in eukaryotes. This review summarizes the recent characterization of the biosynthesis of sulfur modifications in tRNA and the novel roles of this modification in cellular functions in various model organisms, with a special emphasis on 2-thiouridine derivatives. Each biosynthesis pathway of sulfur-containing molecules is mutually modulated via sulfur trafficking, and 2-thiouridine and codon usage bias have been proposed to control the translation of specific genes.

2-Thioribothymidine (s²T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s²T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s²T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s²T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s²T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro.

Thermostability of tRNA in thermophilic bacteria is effected by post-transcriptional modifications, such as 2-thioribothymidine (s²T) at position 54. Using a proteomics approach, we identified two genes (ttuA and ttuB; tRNA-two-thiouridine) that are essential for the synthesis of s²T in Thermus thermophilus. Mutation of either gene completely abolishes thio-modification of s²T, and these mutants exhibit a temperature-sensitive phenotype. These results suggest that bacterial growth at higher temperatures is achieved through the thermal stabilization of tRNA by a 2-thiolation modification. TtuA (TTC0106) is possibly an ATPase possessing a P-loop motif. TtuB (TTC0105) is a putative thio-carrier protein that exhibits significant sequence homology with ThiS of the thiamine synthesis pathway. Both TtuA and TtuB are required for in vitro s²T formation in the presence of cysteine and ATP. The addition of cysteine desulfurases such as IscS (TTC0087) or SufS (TTC1373) enhances the sulfur transfer reaction in vitro.

EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.

2-Thioribothymidine (s²T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s²T54 is known to be responsible for the thermostability of tRNA. The s²T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus. Little is known about the biosynthesis of s²T, including the sulfur donor, modification enzyme, and the tRNA structural requirements. To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNA^Asp with an altered sequence and a host-vector for T. thermophilus. We were able to detectin vivo activity of s²T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization. 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing. To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNA^Aspgene. Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction.