Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation

Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA^Phe, with the natural dipeptidyl fMet-Phe-tRNA^Phe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg²⁺ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.

核糖体转位（ribosomal translocation）的动力学表征对于解析蛋白质合成中的延伸机制至关重要。本研究对停流技术（stopped-flow）下常用的基于荧光标记mRNA的转位实验进行了优化，通过猝灭流法（quench-flow）中的功能性三肽形成实验完成校准。
我们发现，从+1位点起延伸10个碱基的荧光标记mRNA（mRNA+10）最适配两类实验：其形成三肽的速率与更长链的mRNA相当，且在mRNA发生转位时可产生显著的荧光信号变化。
随后，我们在停流实验中对比了常用的肽酰tRNA类似物（peptidyl tRNA analog）N-乙酰苯丙氨酰-tRNAPhe与天然二肽形式的甲酰甲硫氨酰-苯丙氨酰-tRNAPhe的转位行为。结果显示，该类似物的转位速率约为天然二肽tRNA的一半，且呈现双相动力学（biphasic kinetics）特征。当反应温度降低、镁离子（Mg²+）浓度升高时，转位速率会进一步下降；而提升延伸因子G（EF-G）的浓度，则可显著加快转位速率，并大幅提高快相阶段的信号振幅。
综上，本研究建立了一种改进的实时检测方法，可用于监测天然二肽tRNA及其N-乙酰类似物介导的mRNA转位过程。