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Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy

Nature Structural Biology volume 10, pages 899–906 (2003)Cite this article

A Corrigendum to this article was published on 01 December 2003

Abstract

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of ∼9 Å, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.

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Figure 1: Cryo-EM maps showing the incorporation of the aa-tRNA into the ribosomal A site.
Figure 2: Topology of the decoding site.
Figure 3: Ternary complex stalled with kirromycin.
Figure 4: Model of A/T site tRNA and its interaction with ribosomal LH69.
Figure 5: Conformational change of the GAC of the 50S subunit upon interaction of the ternary complex with the ribosome.
Figure 6: Coupling of the conformational change of the GAC with the movement of aa-tRNA.

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Acknowledgements

We thank M. Yarus for a discussion and helpful suggestions and M. Watters for assistance with the illustrations. This work was supported by the Howard Hughes Medical Institute (J.F.), by grants from the US National Institutes of Health (J.F. and S.C.H.) and the US National Science Foundation (J.F.), by the Swedish Foundation for Strategic Research and the Swedish Research Council (A.V.Z. and M.E.), by an Ole Romer research grant from the Danish Research Council and the EMBO Young Investigator Program (P.N.) and by a scholarship from Novo Nordisk/Novozymes (R.N.).

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  1. Mikel Valle

    Present address: Centro Nacional de Biotecnología, CSIC, Universidad Autónoma de Madrid, Cantoblanco, 28049, Madrid, Spain

Authors and Affiliations

  1. Howard Hughes Medical Institute, Health Research, Inc. at the Wadsworth Center, Empire State Plaza, Albany, 12201-0509, New York, USA

    Mikel Valle & Joachim Frank

  2. Department of Cell and Molecular Biology, Biomedical Center, Box 596, Uppsala, S-751 24, Sweden

    Andrey Zavialov & Måns Ehrenberg

  3. Wadsworth Center, Empire State Plaza, Albany, 12201-0509, New York, USA

    Wen Li & Jayati Sengupta

  4. School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, 30332, Georgia, USA

    Scott M Stagg & Stephen C Harvey

  5. Department of Molecular Biology, University of Aarhus, Gustav Wieds vej 10C, Aarhus C, DK-8000, Denmark

    Rikke C Nielsen & Poul Nissen

  6. Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, 12201-0509, New York, USA

    Joachim Frank

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Supplementary information

Supplementary Fig. 1 (PDF 1565 kb)

Supplementary Video 1.

Accommodation process on the 70S ribosome.By alternating between cryoEM maps of (i)the 70S ribosome with the stalled ternary complex (70S ·fMet-tRNA fMet ·Phe-tRNA Phe ·EF-Tu ·GDP ·kir)and (ii)the complex bearing the accommodated A-site tRNA (70S ·tRNA fMet ·MP-tRNA Phe),the dynamic transition during the incorporation of the aa-tRNA can be observed.In the rendering,the segmented and differently colored volumes allow the ribosomal subunits (large subunit in blue,small subunit in yellow)and the tRNAs in P (green)and E sites (orange)to be distinguished. The A/T-and A-site tRNAs are depicted in the same color (magenta),so that the trajectory of the tRNA can be more easily followed during the accommodation. (MOV 2802 kb)

Supplementary Video 2.

tRNA movement and conformational change during accommodation. (MOV 3670 kb)

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Valle, M., Zavialov, A., Li, W. et al. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy. Nat Struct Mol Biol 10, 899–906 (2003). https://doi.org/10.1038/nsb1003

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