Optimization of the translation reaction. (A) Time course of translation reactions assembled with complete CFE (blue) or with ribosomes purified from CFE via 180K-centrifugation (RSR, magenta). Both reactions were programmed with 400 ng of capped TAP-Renilla mRNA reporter; the final reaction volume was 30 µL. Each CFE reaction was estimated to contain 60 µg of the total RNA. To the RSR reactions, we added 24 µg of purified ribosomes. Reaction aliquots (4.5 µL) were collected at the indicated time points, levels of generated reporter proteins were measured by the Renilla luciferase assay, normalized by the 18S rRNA hybridization signal in each sample, and plotted as linear and log10 graphs. Each reaction was set in triplicate. The bottom panel shows a representative northern blot of the RNA extracted from the reactions and hybridized with an 18S rRNA-specific probe. (B,C) Efficiency of translation in RSR assay reactions depends on the concentration of purified ribosomes. (B) Indicated amounts of solubilized ribosomes derived from P180 generated by one-step centrifugation of CFE were added to S180. Each reaction was charged with 200 ng of capped TAP-RLuc mRNA reporter. (C) Total RNA was extracted from the RSR/luciferase reactions from B and analyzed by northern hybridizations using probes specific to 25S rRNA, 18S rRNA, and TAP-RLuc mRNA. (D) mRNA dose dependence. Translation reactions were assembled with complete CFE (blue) or with ribosomes purified from CFE via 180K-centrifugation (RSR, magenta). Reactions were programmed with the indicated amounts of capped TAP-Renilla mRNA reporter; the final reaction volume was 15 µL. For the CFE reaction, we used unfractionated CFE (30 µg total RNA), while the RSR reaction contained 4 µg of purified ribosomes. RNA was extracted from the RSR/luciferase reactions and analyzed by northern hybridization with an 18S rRNA-specific probe (bottom). Radioactive signal corresponding to the full-length 18S rRNA was converted to phosphorimaging units and used to quantify the luminescent signal derived from the same sample. All reactions in B and C were assembled in triplicate and carried out for 90 min at 21°C. In all graphs, error bars represent standard error of the mean (SEM) of three experiments.
