The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin‐related modifier 1 (Urm1) pathway is responsible for the synthesis of 2‐thiolated wobble uridine (U₃₄). During the key step of the modification cascade, the E1‐like activating enzyme ubiquitin‐like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C‐terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1‐COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin‐like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin‐like proteins (UBL) and sulfur‐relay systems. Here, we report the crystal structures of full‐length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C‐terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self‐conjugation with its own product, namely activated Urm1‐COSH.

中文翻译：

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双功能Uba4-Urm1硫中继系统在tRNA硫醇化和泛素样结合中的分子基础。

硫对tRNA碱基的化学修饰对于调节翻译和优化蛋白质合成至关重要。在真核生物中，泛素相关的修饰子1（Urm1）途径负责2硫醇化摆动尿苷的合成（U ₃₄）。在修饰级联反应的关键步骤中，类似E1的活化酶类似泛素的蛋白质活化剂4（Uba4）首先将其底物Urm1的C端进行腺苷酸化和硫代羧化。随后，活化的硫代羧化的Urm1（Urm1-COSH）可以作为特定tRNA硫代酶的硫供体，或参与泛素样的偶联反应。Uba4和Urm1的结构和机理细节仍然难以捉摸，但是对于了解泛素样蛋白（UBL）和硫磺递延系统之间的进化分支点而言，这是关键。在这里，我们报告全长Uba4的晶体结构及其带有底物Urm1的异二聚体复合物。我们展示了Uba4的两个域如何协调Urm1 C末端的识别，结合和硫代羧化。最后，