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Functional epitopes at the ribosome subunit interface

Nature Chemical Biology volume 2, pages 254–258 (2006)Cite this article

Abstract

The ribosome is a 2.5-MDa molecular machine that synthesizes cellular proteins encoded in mRNAs1. The 30S and 50S subunits of the ribosome associate through structurally defined intersubunit bridges2,3,4,5 burying 6,000 Å2, 80% of which is buried in conserved RNA-RNA interactions6. Intersubunit bridges bind translation factors7,8, may coordinate peptide bond formation and translocation9,10,11 and may be actively remodeled in the post-termination complex12,13, but the functional importance of numerous 30S bridge nucleotides had been unknown. We carried out large-scale combinatorial mutagenesis and in vivo selections on 30S nucleotides that form RNA-RNA intersubunit bridges in the Escherichia coli ribosome. We determined the covariation and functional importance of bridge nucleotides, allowing comparison of the structural interface and phylogenetic data to the functional epitope. Our results reveal how information for ribosome function is partitioned across bridges, and suggest a subset of nucleotides that may have measurable effects on individual steps of the translational cycle.

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Figure 1: Selection of functional ribosome intersubunit bridges.
Figure 2: Intersubunit bridges.
Figure 3: Covariation between 16S rRNA intersubunit bridge nucleotide positions.
Figure 4: Functional epitopes in structurally defined intersubunit bridges and interactions between 30S intersubunit bridge nucleotides and 50S.
Figure 5: Function and phylogenetic conservation in intersubunit bridge nucleotides.

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Acknowledgements

J.W.C. is an EMBO Young Investigator. K.W. is grateful for a Medical Research Council–Laboratory of Molecular Biology (MRC-LMB) Cambridge Scholarship, an Honorary External Research Studentship from Trinity College, Cambridge and an Overseas Research Studentship Award. We thank M.E. Hurles (Sanger Institute) for discussions on quantifying sequence polymorphisms, P.H. Dear (LMB) and G. Mitchison (Cambridge) for discussions on statistical methods, and T.M. Schmeing (LMB) and other members of LMB for critically reading versions of the manuscript.

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Author notes

  1. Oliver Rackham and Kaihang Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England, UK

    Oliver Rackham, Kaihang Wang & Jason W Chin

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  1. Oliver Rackham
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  2. Kaihang Wang
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Correspondence to Jason W Chin.

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Supplementary information

Supplementary Table 1

Library design. (PDF 10 kb)

Supplementary Table 2

Mutagenic oligonucleotides for multiposition libraries. (PDF 11 kb)

Supplementary Table 3

Library constructed statistics. (PDF 12 kb)

Supplementary Table 4

Selected sequences. (PDF 35 kb)

Supplementary Table 5

Covariation analysis. (PDF 16 kb)

Supplementary Table 6

Intersubunit bridge selection data and phylogenetic data. (PDF 18 kb)

Supplementary Methods (PDF 49 kb)

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Rackham, O., Wang, K. & Chin, J. Functional epitopes at the ribosome subunit interface. Nat Chem Biol 2, 254–258 (2006). https://doi.org/10.1038/nchembio783

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