Catalyzes the ATP-dependent 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 required for cell growth at high temperatures (PubMed:16547008). TtuA transfers the S atom from the thiocarboxylated C-terminus of TtuB to tRNA (PubMed:28439027). 5-methyluridine(54) in tRNA + [TtuB sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + ATP + H2O = 5-methyl-2-thiouridine(54) in tRNA + [TtuB sulfur-carrier protein]-C-terminal Gly-Gly + AMP + diphosphate + 2 H(+) Binds 1 [4Fe-4S] cluster per subunit. The cluster is chelated by three Cys residues, the Fe with a free coordination site of the Fe-S cluster may contribute to an enzymatic role. Enzymatic activity may be regulated by TtuB conjugation. tRNA modification. Homodimer (PubMed:28439027). Is able to form a heterocomplex with TtuB (PubMed:16547008, PubMed:28439027). Conjugated to TtuB via covalent linkages involving Lys-137, Lys-226 and Lys-229. 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. The thiolation reaction consists of two steps: a first activation step by ATP to form an adenylated intermediate of the target base of tRNA, and a second nucleophilic substitution step of the sulfur (S) atom supplied by thiocarboxylated TtuB for the adenyl group. However, it is not clear if the S-transfer mechanism is direct or indirect. Belongs to the TtcA family. TtuA subfamily.