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High-resolution structure of the Escherichia coli ribosome

Nature Structural & Molecular Biology volume 22, pages 336–341 (2015)Cite this article

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Abstract

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation have remained poorly understood. Moreover, the functions of modifications to ribosomal RNA and ribosomal proteins have also been unclear. Here we present the structure of the Escherichia coli 70S ribosome at 2.4-Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and it suggests how solvation contributes to ribosome integrity and function as well as how the conformation of ribosomal protein uS12 aids in mRNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including post-transcriptional and post-translational modifications, and should serve as a basis for future antibiotic development.

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Figure 1: E. coli 70S ribosome I in an intermediate rotated state.
Figure 2: Solvation at the ribosomal subunit interface and in the nascent peptide–exit tunnel.
Figure 3: Post-transcriptional and post-translational modifications in functional centers of the ribosome.
Figure 4: Pseudouridines and syn-pyrimidines in the ribosome.

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Acknowledgements

The authors wish to thank J. Doudna, A.S.-Y. Lee, A. Pulk and P. Kranzusch for helpful discussions; P. Afonine and P. Adams for advice on the use of feature-enhanced maps; G. Meigs and J. Holton for assistance at beamline 8.3.1 at the Advanced Light Source (ALS); and K. Diederichs for help with XDS. This work was supported by US National Institutes of Health (NIH) grant R01-GM65050 to J.H.D.C.; by NIH grant 2R01GM079238 to S.C.B., D.S.T., M.R.W. and R.B.A.; by the NIH project Macromolecular Insights on Nucleic acids Optimized by Scattering (MINOS), grant R01GM105404, for beamline 8.3.1 at the ALS; and by the US Department of Energy, grant DEAC02-05CH11231, for beamline 8.3.1 at the ALS. J.N. was funded by a Human Frontiers in Science Program Long-Term Postdoctoral Fellowship.

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Jonas Noeske & Jamie H D Cate

  2. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA

    Jonas Noeske & Jamie H D Cate

  3. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA

    Michael R Wasserman, Daniel S Terry, Roger B Altman & Scott C Blanchard

  4. Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medical College, Rockefeller University, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Scott C Blanchard

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  1. Jonas Noeske
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Contributions

J.N. optimized crystal growth and cryostabilization procedures, measured the X-ray diffraction data, solved the structure and carried out refinement and structural analysis. J.H.D.C. assisted with data reduction, refinement and structural analysis. M.R.W., D.S.T., R.B.A. and S.C.B. conducted the smFRET experiments. J.N., J.H.D.C. and S.C.B. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Rotational dynamics of the E. coli ribosome.

(a) E. coli 70S ribosome II in the unrotated state. Ribosomal subunits are colored by atomic displacement factor (ADP) from 20 to 150 Å2. The views are from the perspective of the subunit interface. Features in the 50S subunit include the central protuberance (CP), L1 arm (L1), protein L9 (L9), L7-L12 region (L12), A-site finger (ASF) and the GTPase center (G). In the 30S subunit, these include the head (H), body (B), and platform (PL). (b) Single-molecule imaging of the modulation of tRNA binding states by magnesium ions. Occupancy in classical and hybrid tRNA binding states as measured by smFRET between fluorophores on P- and A-site tRNAs, shown with standard deviations of three technical replicates (Online methods).

Supplementary Figure 2 Feature-enhanced maps of the refined ribosome structure, using structure factors to 2.4-Å or 2.1-Å resolution.

Maps are shown in the vicinity of: (a) β-hydroxy-Arg81 in uL16; (b) D2449 in 23S rRNA; (c) Pro45 in uS12; (d) β-methylthio-Asp89 in uS12; (e) ψ955 in 23S rRNA; (f) U960 in 16S rRNA; and (g) U1779 in 23S rRNA. All maps are contoured at 2.5 standard deviations from the mean, except panel (b) and (d), which are at 2.0 and 2.2 standard deviations from the mean, respectively.

Supplementary Figure 3 Quality of electron density maps in well-ordered regions of the ribosome.

(a) Section of 23S rRNA. (b-d) Different amino acids of ribosomal protein uL2. The feature enhanced maps are contoured at 2.0 standard deviations from the mean. (e) Density consistent with a spermidine, possibly bound in two modes (waters to the right mark extended tube of density), in close proximity to bridge B3. The phosphate of G1935, which would be above the plane, has been removed for clarity. (f) A putrescine molecule bound at the base of H69. In panels e and f, the feature enhanced map is contoured at 2.5 and 2.3 standard deviations from the mean, respectively.

Supplementary Figure 4 Feature-enhanced map of modified nucleotides and amino acids in the ribosome.

(a) Modifications in the 30S subunit, with the map contoured at 1.8 standard deviations from the mean. Amino acid modification in protein uS12 is shown (β-methylthio-Asp81). (b) Modifications in the 50S subunit, with the map contoured at 2.0 standard deviations from the mean. Amino acid modifications in uL3 (N-methyl-Gln150) and uL16 (β-hydroxy-Arg81), respectively, are modeled in alternative conformations, based on the unbiased electron density maps. The arrow points to the β-hydroxy group in β-hydroxy-Arg81. In both panels (a) and (b) the map was generated prior to modeling of the modifications.

Supplementary Figure 5 Solvation of the nascent peptide–exit tunnel.

(a) Antibiotics that bind in the exit tunnel. Shown are erythromycin (white), telithromycine (green), the streptogramin B quinupristin (cyan). (b) View of the constriction site formed by ribosomal proteins uL4 and uL22 in the nascent peptide exit tunnel. An MPD molecule stacks on A751 further constricting the exit tunnel. Feature enhanced map shown at a contour of 2.5 standard deviations from the mean. (c) SecM stalling peptide interactions with the exit tunnel. Stacking of Trp155 of the SecM stalling peptide on A751 of the ribosome, proposed to be critical for ribosome stalling. Panel c is adapted from Gumbart et al.

Supplementary Figure 6 Examples of syn pyrimidines in the ribosome.

(a) U1345 in 16S rRNA forms reverse U-U base pair with U1376. The feature enhanced map is contoured at 2.5 standard deviations from the mean. (b) In 23S rRNA, C1838 in a syn conformation is part of a base triple with A1901 and U1841. The feature enhanced map is contoured at 2.3 standard deviations from the mean.

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Noeske, J., Wasserman, M., Terry, D. et al. High-resolution structure of the Escherichia coli ribosome. Nat Struct Mol Biol 22, 336–341 (2015). https://doi.org/10.1038/nsmb.2994

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