Snapshots of tRNA sulphuration via an adenylated intermediate
Snapshots of tRNA sulphuration via an adenylated intermediate
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DOI：
10.1038/nature04896
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发表时间：
2006-07-27
期刊：
影响因子：
64.8
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
--
作者：
Numata, Tomoyuki;Ikeuchi, Yoshiho;Nureki, Osamu
谷氨酸、赖氨酸和谷氨酰胺转移RNA的第一反密码子位置(U34)的尿苷被硫脲酶普遍修饰为2-硫代尿苷(S2U34)，这是限制核糖体上蛋白质合成过程中密码子-反密码子摆动的精确翻译的关键。然而，目前尚不清楚该酶如何将活性硫结合到尿苷碱基的正确位置上。在这里，我们以三种离散的形式给出了MnmA硫代尿苷酸酶-tRNA复合体的晶体结构，这为RNA硫化过程中的顺序化学反应提供了快照。在酶激活过程中，活性部位上方的α-螺旋被重组为一个特殊的β-发夹结构，该环将翻转的U34深深地包装在催化口袋中，触发催化半胱氨酸残基的激活。腺化的RNA中间体被捕获。因此，络合物的活性闭合构象通过形成一个催化室来防止溶剂进入催化位置，从而确保硫准确地结合到活性尿苷碳中。谷氨酸tRNA络合物的结构进一步揭示了MnmA如何特异性识别其三种不同的tRNA底物。这些发现为酶在生物分子中的精确位置结合反应原子的一般机制提供了结构基础。
Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon–anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase–tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an α-helix overhanging the active site is restructured into an idiosyncratic β-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.