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tRNA selection and kinetic proofreading in translation

Nature Structural & Molecular Biology volume 11, pages 1008–1014 (2004)Cite this article

Abstract

Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu–GTP–aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.

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Figure 1: Single-molecule fluorescence intensity and FRET time traces.
Figure 2: Contour plots of the time evolution of population FRET.
Figure 3: A revised model for tRNA selection at the A site inferred from smFRET data (based on the scheme described in ref. 17) schematizing EF-Tu-regulated movements of aa-tRNA into the accommodated state (0.75 FRET).

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Acknowledgements

S.C.B. is supported by the Giannini Family Foundation, and R.L.G. is supported by the American Cancer Society. Restrictocin was a gift of C. Correll (University of Chicago). This work was supported by grants to J.D.P. from the US National Institutes of Health (GM51266) and the David and Lucille Packard Foundation, to S.C. from the US National Science Foundation, the US National Aeronautics and Space Administration and the US Air Force Office of Scientific Research, and to S.C. and J.D.P. from the David and Lucille Packard Foundation Interdisciplinary Science Program (grant 2000-01671). The authors thank E. Lau for technical support, J. Hoch for suggestions relating to fluorescence quenching, E.V. Puglisi for critical discussions and scientific input, and M. Dorywalska and J. Choy for comments on the written manuscript.

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Author notes

  1. Ruben L Gonzalez and Harold D Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics and Applied Physics, Stanford University, Stanford, 94305-4060, California, USA

    Scott C Blanchard, Harold D Kim & Steven Chu

  2. Department of Structural Biology, Stanford University School of Medicine, Stanford, 94305-5126, California, USA

    Scott C Blanchard, Ruben L Gonzalez Jr & Joseph D Puglisi

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Correspondence to Steven Chu or Joseph D Puglisi.

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Supplementary information

Supplementary Fig. 1

Histogram analysis of FRET states achieved after EF-Tu(GTP)Phe-tRNAPhe delivery to surface immobilized ribosome complexes. (PDF 58 kb)

Supplementary Fig. 2

FRET arrival time plot. (PDF 58 kb)

Supplementary Fig. 3

Time evolution of FRET states in the delivery of EF-Tu(GTP)Phe-tRNAPhe (Cy5-acp3U) to surface immobilized 70S complexes initiated with fMet-tRNAfMet (Cy3-s4U) in the P site and a phenylalanine codon (UUU) in the A site. (PDF 58 kb)

Supplementary Fig. 4

Lifetime analysis of the 0.35 FRET state in tetracycline-stalled EF-Tu(GTP)Phe-tRNAPhe delivery to the ribosome. (PDF 36 kb)

Supplementary Fig. 5

The lifetime of the 0.5 FRET state (pre-GTP hydrolysis). GDPNP is a non-hydrolyzable GTP analogue. (PDF 370 kb)

Supplementary Fig. 6

Cleavage of the SRL within tight coupled-70S particles was achieved using the enzyme restrictocin. (PDF 114 kb)

Supplementary Fig. 7

EF-Tu(GTP)Phe-tRNAPhe (Cy5-acp3U) is stalled in the 0.5 FRET state when delivered to immobilized ribosome complexes cleaved at the SRL. (PDF 29 kb)

Supplementary Fig. 8

Lifetime analysis of the 0.35 FRET state in delivery of EF-Tu(GDPNP)Phe-tRNAPhe to ribosome complexes programmed with a cognate (UUU). (PDF 29 kb)

Supplementary Fig. 9

Lifetime analysis of the 0.35 FRET state prior to transition to the zero FRET state in delivery of EF-Tu(GTP)Phe-tRNAPhe to ribosome complexes programmed with a cognate (UUU) and near-cognate (CUU) codon. (PDF 32 kb)

Supplementary Table 1

Fidelity calculations. (PDF 25 kb)

Supplementary Methods (PDF 54 kb)

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Blanchard, S., Gonzalez, R., Kim, H. et al. tRNA selection and kinetic proofreading in translation. Nat Struct Mol Biol 11, 1008–1014 (2004). https://doi.org/10.1038/nsmb831

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