Ikeuchi Yoshiho, Shigi Naoki, Kato Jun-Ichi, Nishimura Akiko, Suzuki Tsutomu
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Mol Cell. 2006 Jan 6;21(1):97-108. doi: 10.1016/j.molcel.2005.11.001.
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.
负责NNR密码子的细菌tRNA的摆动碱基被修饰为5-甲基氨基甲基-2-硫代尿苷（mnm5s2U）。mnm5s2U的2-硫代修饰对于准确解码是必需的，并且对正常细胞生长至关重要。我们通过全基因组系统筛选（“核糖核酸组分析”）鉴定出五个基因yhhP、yheL、yheM、yheN和yccK（分别命名为tusA、tusB、tusC、tusD和tusE），它们对于mnm5s2U的2-硫代尿苷化是必需的。用重组TusA、TusBCD复合物、TusE以及先前鉴定的IscS和MnmA在体外重建了高效的2-硫代尿苷形成。IscS的脱硫酶活性受到TusA结合的刺激。IscS将过硫化物硫转移给TusA。TusE结合TusBCD复合物并刺激硫从TusA转移到TusD。TusE还与MnmA-tRNA复合物相互作用。这项研究表明，2-硫代尿苷的形成通过一个复杂的硫传递系统进行，该系统由多种硫介质组成，这些介质选择并促进特定的硫流从各种硫运输途径流向2-硫代尿苷。
