Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions.
The wobble bases of bacterial tRNAs responsible for NNR codons are modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio modification of mnm5s2U is required for accurate decoding and essential for normal cell growth. We identified five genes yhhP, yheL, yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE, respectively) that are essential for 2-thiouridylation of mnm5s2U by a systematic genome-wide screen ("ribonucleome analysis"). Efficient 2-thiouridine formation in vitro was reconstituted with recombinant TusA, a TusBCD complex, TusE, and previously identified IscS and MnmA. The desulfurase activity of IscS is stimulated by TusA binding. IscS transfers the persulfide sulfur to TusA. TusE binds TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE also interacts with an MnmA-tRNA complex. This study revealed that 2-thiouridine formation proceeds through a complex sulfur-relay system composed of multiple sulfur mediators that select and facilitate specific sulfur flow to 2-thiouridine from various pathways of sulfur trafficking.
Ikeuchi
Yoshiho
Y
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Shigi
Naoki
N
Kato
Jun-Ichi
J
Nishimura
Akiko
A
Suzuki
Tsutomu
T
D000595:Amino Acid Sequence; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013455:Sulfur / Q000378:metabolism*; D013891:Thiouridine / Q000378:metabolism*
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D011994:Recombinant Proteins; C489236:tus protein, E coli; D013891:Thiouridine; D013455:Sulfur; D012343:RNA, Transfer; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase