Plays a central role in 2-thiolation of mcm(5)S(2)U at tRNA wobble positions of tRNA(Lys), tRNA(Glu) and tRNA(Gln). Directly binds tRNAs and probably acts by catalyzing adenylation of tRNAs, an intermediate required for 2-thiolation. It is unclear whether it acts as a sulfurtransferase that transfers sulfur from thiocarboxylated URM1 onto the uridine of tRNAs at wobble position. {ECO:0000255|HAMAP- Rule:MF_03053, ECO:0000269|PubMed:19017811}. This is the function of CTU1 (cytosolic thiouridylase subunit 1, Ensembl gene identifier ENSG00000142544).