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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Nature. 2010 Apr 15;464(7291):1012–1017. doi: 10.1038/nature08925

Figure 3. Real-time translation at near physiological concentrations.

Figure 3

a. Two heteropolymeric mRNAs encoding 13 amino acids were used: M(FK)6 and M(FKK)4. Translation was observed in the presence of 200 nM Phe-(Cy5)tRNAPhe, 200 nM Lys-(Cy2)tRNALys TC and 500 nM EF-G as a series of fluorescent pulses that mirror the mRNA sequence. A long Cy2 pulse is observed upon arrival of the ribosome at the stop codon. Brief sampling pulses (<100 ms) of both Lys-(Cy2)tRNALys and Phe-(Cy5)tRNAPhe TC are observed after arrival at the stop codon. b. Event histograms for translation of M(FK)6 showing translation out to 12 elongation codons (red, n=381). In the presence of 1 μM erythromycin, translation (blue, n=201) is stalled at codon 8 of the mRNA. c. Analysis of translation rates at each codon in M(FK)6. Mean times (avg. ± s. d.) between tRNA arrival events are plotted for translation in the presence of 200nM TC and 30, 100 and 500 nM EF-G. d. Overall tRNA transit times (avg. ± s. d.) for codons 2-12 at (from left) 200 nM TC and 30, 100, or 200 nM EF-G; 500 nM TC and 500 nM EF-G; and 200 nM TC/500 nM EF-G in the presence of 1 μM fusidic acid. e. Cumulative translation times (avg. ± s. d.) for each codon in M(FK)6 at 200 nM TC and 30, 100, or 200 nM EF-G; 500 nM TC and 500 nM EF-G, and 200 nM TC/500 nM EF-G in the presence of 1 μM fusidic acid.