Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions.
Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions.
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DOI：
10.1093/nar/gkn1023
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发表时间：
2009-03
影响因子：
14.9
通讯作者：
Suzuki T
中科院分区：
文献类型：
--
作者：
Noma A;Sakaguchi Y;Suzuki T
trna中的摆动修饰，5-甲氧基羰基甲基-2-硫脲（mcm5s2U），是真核生物正确解码NNR密码子所必需的。2-硫基团通过在很大程度上固定C3 ' -内切核糖皱缩，赋予mcm5s2U构象刚性，确保稳定和准确的密码子-反密码子配对。我们在酿酒酵母中鉴定出5个基因，YIL008w （URM1）、YHR111w （UBA4）、YOR251c （TUM1）、YNL119w （NCS2）和YGL211w (NCS6)，它们是mcm5s2U 2-硫基化所必需的。体外硫转移实验表明，Tum1p刺激Nfs1p的半胱氨酸脱硫酶，并接受Nfs1p的过硫硫。URM1是一种与泛素相关的修饰剂，而UBA4是一种类似e1的酶，参与蛋白质的泛素化。Urm1p的羧基端被激活为酰基腺苷酸（-COAMP），然后被Uba4p激活为硫代羧化（-COSH）。活化的硫代羧酸盐可用于后续由Ncs2p/Ncs6p介导的2-硫脲生成反应。我们可以利用重组蛋白在体外成功地重建2-硫脲的结构。本研究揭示了2-硫脲的形成与蛋白脲基化具有相同的途径和化学反应。真核2-硫脲形成的硫流机制不同于细菌的过硫化学硫接力系统。
The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm5s2U by largely fixing the C3′-endo ribose puckering, ensuring stable and accurate codon–anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm5s2U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.
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