Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system

Abstract

The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the "initiation potential" of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5'-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation.

Figure 1. Time course of the synthesis and accumulation of functionally active luciferase in the cell-free translation reactor.

The translation was started and programmed by the addition of Luc-mRNA up to its final concentration of 50 pmol per ml. (a) The time course curve of the luciferase synthesis. Three stages can be noted: (1) the initial lag with duration of about 8–9 min (this time corresponds to the transit time of the moving ribosome along the mRNA), when no complete active proteins appeared yet; (2) the productive period of the active protein synthesis during ca. 20 min; and (3) the period of the synthesis deceleration passing into plateau. (b) Time course of the accumulation of functionally active luciferase in the translation reactor. The translation system was initially started by the addition of Luc-mRNA up to its final concentration of 50 pmol per ml. The capped free Luc-mRNA was added into the active translation system (“post-start mRNA addition”) up to the final concentration of 50 pmol per ml during the productive protein synthesis stage, just before the plateau stage, as marked by vertical arrow. (c) All was the same as in Fig. 1b, but total transfer RNA was added as a control to the translating system instead of mRNA.

Figure 2. Time courses of the synthesis of functionally active luciferase (left) and Green Fluorescent Protein (GFP, right) in the wheat germ cell-free translation system.

The translation was started and programmed by Luc-mRNA (the final concentration of 50 pmol/ml). The GFP mRNA was added into the active translation system just before the plateau stage as marked by vertical arrow up to the final concentration of 400 pmol/ml (red curve). No mRNA was added in the case of the parallel control translation reaction (blue curve). Note that in response to the addition of the GFP mRNA the rise of luciferase luminescence was observed (see red curve after the arrow). In 10 min after the GFP mRNA addition, aliquots were taken to measure fluorescence of the synthesized GFP (green curve).

Figure 3. Dependence of the protein release stimulation effect on the added free mRNA concentration and on the presence of the cap structure.

(a,b) The translation system was started and run as described in Fig. 1 legend. (a) The free uncapped Luc-mRNA with omega leader sequence (see Methods section) was added to the translation mixture at the time before the plateau, the added mRNA final concentrations being adjusted up to 50 pmol/ml (cyan curve), 100 pmol/ml (blue curve) and 250 pmol/ml (red curve) in three parallel translation runs, respectively. (b) The capped (green curve) and uncapped (black curve) Luc-mRNAs with β-globin leader sequence were added to the translation system up to their final concentration of 50 pmol/ml, during the active protein (luciferase) synthesis stage, at the time point before the plateau stage, as described in Fig. 1b legend. The time point of the post-start additions of the free mRNA is marked by vertical arrows.

Figure 4. Dependence of the protein release stimulation effect on the presence of the stop codon in the primary template mRNA of the cell-free system.

Note that the release of the active protein upon the post-start addition of free mRNA was found to be insignificant in the case of the mRNA deprived of the stop codon. (a,b) The translation system was started by the addition of the Luc-mRNA up to its final concentration of 50 pmol/ml (red curves), and the same mRNA in the same concentration, but deprived of the stop codon (blue curves). The free Luc-mRNA was added to the translation mixture up to its final concentrations of 100 pmol/ml (a) and 250 pmol/ml (b). The time points of the post-start mRNA additions are marked by vertical arrows.