TtuA and TtuB (TTC0105), together with TtuC (TTC0979) and cysteine desulfurases (IscS = TTC0087 or SufS = TTC1373) are responsible for m5s2U (s2T) formation in tRNA of hyperthermophilic bacteria. The reaction requires cysteine (as a sulfur source) and ATP. The C-terminal glycine of TtuB is first activated as an acyl-adenylate by TtuC and then thiocarboxylated by cysteine desulfurases. The sulfur atom of thiocarboxylated TtuB is transferred to tRNA by TtuA. TtuB is a thio-carrier protein that exhibits significant sequence similarity with ThiS of the thiamine synthesis pathway. There may exist additional factors required in the last step of m5s2U biosynthesis because sulfur transfer from TtuB-COSH to tRNA in vitro depends on the addition of a cell extract and the efficiency of tRNA thiolation is low. The activity of the TtuA-C proteins was tested in vitro on substrate yeast tRNAPhe having a m5s2U54. TtuC, an ATPase, seems also to be involved in the biosynthesis of molybdenum cofactor and thiamin.