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Following translation by single ribosomes one codon at a time

Abstract

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

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Figure 1: Experimental design.
Figure 2: Codon-by-codon translation of VE60hp.
Figure 3: Translational trajectories and arrests for VE274hp.
Figure 4: Dwell times and force effect.

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Acknowledgements

We thank R. Hanna for her early efforts on this project, and S. B. Smith for help with the instrumentation. The work was supported by National Institutes of Health grants (to I.T., C.B. and H.F.N.), and a Grant-in-Aid for Young Scientists (A) from the Japan Society for the Promotion of Science (S.Y.).

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Correspondence to Ignacio Tinoco.

Supplementary information

Supplementary Information

The file contains Supplementary Discussion and Supplementary Figures S1 – S7 with Legends. The Supplementary Discussion shows kinetic models and translation with different mRNAs. The Supplementary Figures show force-extension curves at different translation progress for different hairpins, SDS-PAGE gels from bulk translation, distribution of dwell times, and translation rates under various conditions (PDF 487 kb)

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Wen, JD., Lancaster, L., Hodges, C. et al. Following translation by single ribosomes one codon at a time. Nature 452, 598–603 (2008). https://doi.org/10.1038/nature06716

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