Required for the 2-thiolation of 5-methyluridine residue at position 54 in the T loop of tRNAs, leading to 5-methyl-2-thiouridine (m(5)s(2)U or s(2)T) (PubMed:16547008, PubMed:28439027). This modification allows thermal stabilization of tRNAs in thermophilic microorganisms, and is essential for cell growth at high temperatures (PubMed:16547008). Thiocarboxylated TtuB functions as the sulfur donor in the sulfurtransferase reaction catalyzed by TtuA (PubMed:28439027, PubMed:19037260). TtuB also functions as a protein modifier covalently attached to lysine residues of the target proteins TtuA and TtuC (PubMed:22467871). TtuB conjugation might play a regulatory role to ensure appropriate sulfur transfer in cells (PubMed:22467871). tRNA modification. Is able to form a heterocomplex with TtuA. The C-terminal glycine residue of TtuB is first activated by TtuC as an acyl-adenylate (TtuB-COAMP), and then converted to the thiocarboxylate form (TtuB-COSH) by the cysteine desulfurases IscS or SufS. Disruption of this gene completely abolishes the presence of m(5)s(2)U in tRNAs, although its precursor, 5-methyluridine (ribothymidine) is present; other nucleoside modifications remain unchanged. These deletion mutants exhibit a temperature-sensitive phenotype, they are unable to grow above 80 degrees Celsius. Belongs to the TtuB family.