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Unusual base pairing during the decoding of a stop codon by the ribosome

Abstract

During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine–purine base pairs1. Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNASer bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine–purine base pairs at the second and third positions of the codon, which show an unusual Watson–Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs.

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Figure 1: Chemical differences between uridine and pseudouridine, and experimental set-up.
Figure 2: Overall and detailed view of the base pairs involved in the codon–anticodon interaction.
Figure 3: Interaction of ribosomal bases with the codon–anticodon base pairs.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the PDB under accessions 4JV5, 4JYA, 4K0K (30S) and 4K0L, 4K0M, 4K0P, 4K0Q (70S).

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Acknowledgements

We thank D. Hall and G. Winter for help and advice with data collection at beamline I04, Diamond Light Source; T. Tomizaki at beamline X06SA for help with data collection at the Swiss Light Source; and A. McCarthy at beamline ID14-4, ESRF, where screening and initial data collection were done. We thank M. Härtlein for the gift of an overproducing tRNASer clone, M. Torrent for advice on yeast tRNA abundance, and G. Murshudov for advice and help with data analysis and refinement. V.R. was supported by the UK Medical Research Council (grant U105184332), a Programme Grant and Senior Investigator Award from the Wellcome Trust, the Agouron Institute and the Louis-Jeantet Foundation. Y.-T.Y. was supported by a grant from the National Institutes of Health (GM104077), and by the University of Rochester CTSA award (UL1TR000042) from the National Center for Advancing Translational Sciences of the National Institutes of Health. I.S.F. was supported by a postdoctoral fellowship from the Fundacion Ramon Areces.

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Contributions

I.S.F. carried out the crystallographic experiments and analysis and helped write the paper, G.W. did the in vitro translation assays, C.L.N. helped with crystallographic data collection, A.C.K. made the 30S subunits, 70S ribosome and tRNASer, and Y.-T.Y. and V.R. oversaw the project and helped write the paper.

Corresponding authors

Correspondence to Yi-Tao Yu or V. Ramakrishnan.

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The authors declare no competing financial interests.

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Fernández, I., Ng, C., Kelley, A. et al. Unusual base pairing during the decoding of a stop codon by the ribosome. Nature 500, 107–110 (2013). https://doi.org/10.1038/nature12302

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