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

초록

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. This study presents a strategy for the efficient generation of Fluo-Met-tRNAiMet, a fluorescent methionyl initiator tRNA, in living cells. The approach involves the formation of the PFM-tRNAiMet complex using purified fluorescent methionine (PFM), which effectively enables highly chromogenic N-terminal labeling.In addition, conjugation of PFM with cellular tRNAiMet allows the generation of Fluo-Met-tRNAiMet complexes in living cells. Consequent ratiometric fluorescence assays show that PFM-cellular tRNAiMet complexes exhibit higher fluorescence compared to non-PFM complexes. image

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