Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.