| Comments |
P.979 right column bottom paragraph: "Next, [investigators] measured the translocation speed of ribosomes on single mRNAs by treating cells with harringtonine, a small molecule inhibitor of translation that stalls new ribosomes at the start of the mRNA coding sequence without affecting ribosomes further downstream (Ingolia et al., 2011). As mRNA-bound ribosomes complete translation one-by-one after harringtonine treatment, the GFP signal on mRNAs decreases (Figures 2B–2D Movie S4). Using a simple mathematical model to fit the decay in fluorescence of a cumulative curve from many mRNAs (Figure S7 Supplemental Experimental Procedures), [they] estimate a ribosome translocation rate of 3.5 ± 1.1 codons/s. In a parallel approach, [they] also measured the total time required for runoff of all ribosomes from individual mRNAs (Figure S2E), from which [they] calculated a similar translation elongation rate (3.1 ± 0.14 codons/s) as the one obtained through [their] model (Supplemental Experimental Procedures). A reporter with only 5 instead of 24 SunTag peptides showed similar elongation kinetics (3.1 ± 0.4 codons/s) (Figure S2F), indicating that translocation rates are likely not affected by SunTag labeling of the nascent chain. Finally, [they] measured elongation rates of a shorter and codon-optimized reporter gene, which revealed a somewhat faster elongation rate of 4.9 codons/s (Figure S2G), indicating that elongation rates may differ on different transcripts. Using the elongation rate and ribosome density described above, [they] were able to estimate the translation initiation rate to be between 1.4–3.6 min^−1 on the Kif18b reporter (Supplemental Experimental Procedures)." |