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Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome

Nature Structural & Molecular Biology volume 14, pages 30–36 (2007)Cite this article

Abstract

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.

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Figure 1: Role of conserved decoding-site nucleotides in cognate tRNA selection.
Figure 2: Effect of aminoglycosides on the decoding-site variant ribosomes.
Figure 3: Effect of a miscoding tRNA variant on the decoding-site variant ribosomes.
Figure 4: Scheme for a two-barrier model for tRNA selection.

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Acknowledgements

We thank E. Youngman for developing the ribosome tagging and purification methodology, K. Gromadski and M. Rodnina for their technical advice, J. Lorsch, C. Merryman and E. Youngman for their comments and suggestions on the manuscript and O. Uhlenbeck, V. Ramakrishnan and G. Seydoux for valuable comments and discussions. We also thank the US National Institutes of Health for funding of the project and the Howard Hughes Medical Institute for salary support to R.G.

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  1. Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Luisa Cochella, Julie L Brunelle & Rachel Green

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  1. Luisa Cochella
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Correspondence to Rachel Green.

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Supplementary information

Supplementary Fig. 1

Saturating ribosome concentration in GTP hydrolysis reactions. (PDF 48 kb)

Supplementary Fig. 2

Dipeptide formation with excess ternary complex relative to ribosomes. (PDF 58 kb)

Supplementary Fig. 3

Saturating paromomycin concentration in dipeptide formation reactions. (PDF 48 kb)

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Cochella, L., Brunelle, J. & Green, R. Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome. Nat Struct Mol Biol 14, 30–36 (2007). https://doi.org/10.1038/nsmb1183

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