Abstract
Eukaryotic mRNAs are predominantly translated via the cap-dependent pathway. Initiation is a rate-limiting step in cap-dependent translation and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for characterizing the cap-dependent initiation kinetics. Here, we report an in vitro single-molecule assay that allows characterization of both initiation and peptide chain elongation kinetics for cap-dependent translation. Surprisingly, the histogram of the first-round initiation time is highly asymmetrical and spans a large time range that is several-fold greater than the average peptide synthesis time in translation reactions with a firefly luciferase-encoding mRNA. Both the histogram and single-molecule trajectories reveal an unexpected high-degree of asynchrony in translation activity between mRNA molecules. Furthermore, by inserting a small stem-loop (ΔG = −4.8 kcal/mol) in the middle of the mRNA 5′ untranslated region (UTR), our assay robustly detects small changes in budding yeast initiation kinetics, which could not be resolved by bulk luminescence kinetics. Lastly, we demonstrate the general applicability of this assay to distinct cell-free translation systems by using extracts prepared from budding yeast, wheat germ, and rabbit reticulocyte lysates. This assay should facilitate mechanistic studies of eukaryotic cap-dependent translation initiation and translational control.
Highlights
Cap-dependent translation is the predominant pathway for eukaryotic translation [1,2,3,4]
We demonstrated a single-molecule strategy for characterizing in vitro eukaryotic cap-dependent translation initiation kinetics by measuring Cy3-antiFLAG binding to nascent N-terminal 3xFLAG peptides during active translation
Our standard experimental conditions worked well with yeast translation extract (YE), Rabbit Reticulocyte Lysate (RRL) and Wheat germ extract (WGE), and WGE functioned well even under a condition that is more prone to nonspecific binding
Summary
Cap-dependent translation is the predominant pathway for eukaryotic translation [1,2,3,4]. Various approaches were developed for measuring the overall progression of the translation process, including luciferase- [11,12] and SNAP-based [13] assays. These approaches all detect the synthesis of large protein products. Due to the high fluorescent background in cells, an mRNA engaged in active translation was detected when bound with multiple antibodies. All these existing approaches lack high resolution for measuring the kinetics of individual initiation events, the average initiation rate can often be estimated from the experimental observables by mathematical modeling
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