MnmA and IscS Are Required for in Vitro 2-Thiouridine Biosynthesis in Escherichia coli

Abstract

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.

The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity

“…Beside the already mentioned MnmA thiolase, six other proteins were required, and among those only the cysteine desulfurase is clearly conserved in eukaryotes. From this study and others (12,24) it appeared that the thiolase is the final acceptor of a sulfur flow and recognizes tRNAs while the PP-loop activates the C2 position of the uracil ring at position 34 by forming an adenylate intermediate. We reasoned that combining TAP purifications of both Ctu1-TAP and Nfs1-TAP (the fission yeast cysteine desulfurase) might allow in vitro tRNA LYS thiolation detectable by APM/PAGE.…”

“…In eukaryotes, the mitochondrial thiolase Mtu1 is clearly related to the bacterial thiolase MnmA (13,24), but, strikingly, it appears that its cytosolic counterpart, Ctu1, which we have identified in this study, is much more closely related to another bacterial thiolase, TtcA (6), which is involved in synthesis of 2-thiocytidine at position 32 in tRNAs, a modification absent in eukaryotes (see also the Introduction and ref. 14).…”

Catalytic Mechanism of Sep-tRNA:Cys-tRNA Synthase

“…SepCysS Forms Disulfide Linkage-Intramolecular disulfide bonds are often formed between the sulfane sulfur-carrying Cys and a second catalytic Cys as a result of sulfur donation by sulfur transfer enzymes, such as ThiI (23,24) and MnmA (25,26). Two methods were used to determine whether SepCysS forms disulfide bonds.…”

MiaB Protein Is a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in Thiolation and Methylation of tRNA

“…The Source of the Sulfur Atom-The source of the sulfur atom present in thionucleosides has been previously investigated using whole cells (31,32), cell-free (7), or purified systems (33)(34)(35). Cells growing in the presence of [ 35 S]cysteine generate 35 S-labeled ms 2 i 6 A demonstrating that the primary source of the sulfur atom in ms 2 i 6 A is cysteine (36,37).…”