Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA^Phe that are in perfect agreement with previous indirect estimates, and once fMet-tRNA^fMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

我们将试管生化实验的结果直接与活细胞动力学联系起来的能力非常有限。在这里，我们目前提供实验和分析工具，以直接研究活细胞中快速生化反应的动力学。将染料标记的分子电穿孔到细菌细胞中，并使用超分辨单分子显微镜进行跟踪。轨迹通过机器学习算法进行分析，以直接监视绑定状态和自由状态之间的转换。特别是，我们测量了tRNA在核糖体上的停留时间，从而以密码子分辨率实现了活细胞内翻译速率的直接测量。我们发现tRNA ^Phe的延伸率与先前的间接估计完全吻合，并且一旦fMet-tRNA ^fMet由于已与30S核糖体亚基结合，翻译起始速度出奇地快，并且不限制蛋白质合成的整体速度。用于在活细胞中进行直接动力学测量的实验和分析工具的应用远远超出了细菌蛋白质的合成。