Highly chromophoric fluorescent‐labeled methionyl‐initiator tRNAs applicable in living cells

Abstract

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.