Fluorescent initiator tRNAs (tRNAi) play a crucial role in studying protein synthesis, yet generating highly fluorescent tRNAi complexes remains challenging. We present an optimized strategy to effectively generate highly fluorescent initiator‐tRNA complexes in living cells. Our strategy allows the generation of Fluo‐Met‐tRNAiMet complexes. These complexes can have highly chromogenic N‐terminal labeling. For generating such complexes, we use either purified fluorescent methionine (PFM) or non‐purified fluorescently labeled methionine (NPFM). Furthermore, PFM promotes the active generation of endogenous tRNAi in cells, leading to highly efficient Fluo‐Met‐tRNAiMet complexes. Finally, PFM‐tRNAiMet complexes also facilitate the visualization of native fluorescently labeled Tat binding to beads. This demonstrates the potential of our approach to advance precision protein engineering and biotechnology applications.

中文翻译：

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适用于活细胞的高度发色荧光标记的甲硫氨酰引发剂 tRNA

荧光引发剂 tRNA (tRNAi) 在研究蛋白质合成中发挥着至关重要的作用，但生成高荧光 tRNAi 复合物仍然具有挑战性。我们提出了一种优化策略，可在活细胞中有效生成高荧光引发剂-tRNA 复合物。我们的策略允许生成 Fluo-Met-tRNAi蛋氨酸复合物。这些复合物可以具有高度显色的 N 末端标记。为了生成此类复合物，我们使用纯化的荧光蛋氨酸（PFM）或未纯化的荧光标记蛋氨酸（NPFM）。此外，PFM 促进细胞内源性 tRNAi 的活跃生成，从而产生高效的 Fluo-Met-tRNAi蛋氨酸复合物。最后，PFM-tRNAi蛋氨酸复合物还有助于观察天然荧光标记的 Tat 与珠子的结合。这证明了我们的方法在推进精密蛋白质工程和生物技术应用方面的潜力。