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Real-time tRNA transit on single translating ribosomes at codon resolution

Abstract

Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)—labelled with distinct fluorophores—to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

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Figure 1: Translation in ZMWs.
Figure 2: Monitoring translation by fluorescent tRNA-binding events.
Figure 3: Real-time translation at near-physiological concentrations.
Figure 4: A-site sampling on ribosomes stalled at the stop codon.
Figure 5: Monitoring the dynamic tRNA occupancy of translating ribosomes.

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Acknowledgements

This work was supported by National Institutes of Health grant GM51266 (to J.D.P.). We thank T. Funatsu, A. Tsai and A. Petrov for encouragement and discussions. We thank J. Gray for performing ellipsometry experiments.

Author Contributions S.U. performed all experiments and data analysis. S.U., C.E.A. and J.D.P. discussed results and wrote the manuscript. S.W.T., J.K. and B.A.F. provided technical expertise with instrumentation and data processing.

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Correspondence to Joseph D. Puglisi.

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S.W.T., B.A.F. and J.K. are employees and stock option holders, and J.D.P. a consultant, of Pacific Biosciences, a company commercializing sequencing technologies.

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Uemura, S., Aitken, C., Korlach, J. et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010). https://doi.org/10.1038/nature08925

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