Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3′-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3′-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.

中文翻译：

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结核分枝杆菌 tRNA 末端核苷酸转移酶毒素 MenT3 的底物特异性

结核分枝杆菌转移 RNA (tRNA) 末端核苷酸转移酶毒素 MenT3 在 tRNA 的 3'-CCA 末端掺入核苷酸，阻断其氨酰化并抑制蛋白质合成。在这里，我们证明 MenT3 最有效地将 CMP 添加到 tRNA 的 3'-CCA 末端。MenT3 与 CTP 复合物的晶体结构揭示了 CTP 特异性核苷酸结合口袋。CTP中的4-NH2和胞嘧啶的N3和O2原子分别与P120的主链羰基氧和R238的侧链形成氢键。MenT3 在大肠杆菌中的表达选择性降低丝氨酰-tRNASers 的水平，表明 MenT3 对 tRNASers 的特异性失活。一致的是，在测试的大肠杆菌 tRNA 物种中，MenT3 将 CMP 整合到 tRNASer 中的效率最高。II 类 tRNASers 特有的较长可变环对于 MenT3 有效地将 CMP 掺入 tRNASer 中至关重要。用结核分枝杆菌 tRNASer 的较长可变环替换大肠杆菌 tRNAAla 的可变环，使 MenT3 能够将 CMP 整合到嵌合 tRNAAla 中。MenT3 的 N 端带正电区域是 CMP 掺入 tRNASer 所必需的。tRNA 与 MenT3 的对接模型表明，N 端区域与 tRNASer 的较长可变环之间的相互作用促进了 tRNA 底物选择。