Catalyzes the 2-thiolation of uridine at the wobble position (U34) of tRNA(Lys), tRNA(Glu) and tRNA(Gln), leading to the formation of s(2)U34, the first step of tRNA-mnm(5)s(2)U34 synthesis. Sulfur is provided by IscS, via a sulfur-relay system. Binds ATP and its substrate tRNAs. AH2 + ATP + S-sulfanyl-L-cysteinyl-[protein] + uridine(34) in tRNA = 2-thiouridine(34) in tRNA + A + AMP + diphosphate + H(+) + L-cysteinyl-[protein] Interacts with TusE. During the reaction, ATP is used to activate the C2 atom of U34 by adenylation. After this, the persulfide sulfur on the catalytic cysteine is transferred to the C2 atom of U34. The reaction probably involves hydrogen sulfide that is generated from the persulfide intermediate and that acts as nucleophile towards the activated C2 atom on U34. Subsequently, Cys-102 acts as nucleophile towards Cys-199, and a transient disulfide bond is formed. Belongs to the MnmA/TRMU family. Was originally thought to be a 5-methylaminomethyl-2-methyltransferase involved in tRNA modification.