Three-dimensional structure-guided evolution of a ribosome with tethered subunits (original) (raw)

Data availability

The authors declare that all experimental data supporting the findings of this study are available within the paper and its supplementary files. Publicly available data, such as the 4YBB (PDB) ribosome structure are mentioned explicitly when used. All data related to models are available upon request from the authors. The map and fitted model for the cryo-EM data are reported as Electron Microscopy Data Bank entry no. EMD-26666 and PDB structure 7UPH. Source data are provided with this paper.

Code availability

All inputs and command files used in setting up computational modeling are available at https://github.com/everyday847/ribotv3_simulations.

References

  1. Dedkova, L. M., Fahmi, N. E., Golovine, S. Y. & Hecht, S. M. Enhanced d-amino acid incorporation into protein by modified ribosomes. J. Am. Chem. Soc. 125, 6616–6617 (2003).
    Article CAS PubMed Google Scholar
  2. Dedkova, L. M., Fahmi, N. E., Golovine, S. Y. & Hecht, S. M. Construction of modified ribosomes for incorporation of d-amino acids into proteins. Biochemistry 45, 15541–15551 (2006).
    Article CAS PubMed Google Scholar
  3. Dedkova, L. M. et al. β-Puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids. Biochemistry 51, 401–415 (2012).
    Article CAS PubMed Google Scholar
  4. Dedkova, L. M. & Hecht, S. M. Expanding the scope of protein synthesis using modified ribosomes. J. Am. Chem. Soc. 141, 6430–6447 (2019).
    Article CAS PubMed PubMed Central Google Scholar
  5. Des Soye, B. J., Patel, J. R., Isaacs, F. J. & Jewett, M. C. Repurposing the translation apparatus for synthetic biology. Curr. Opin. Chem. Biol. 28, 83–90 (2015).
    Article CAS Google Scholar
  6. Ellefson, J. W. et al. Synthetic evolutionary origin of a proofreading reverse transcriptase. Science 352, 1590–1593 (2016).
    Article CAS PubMed Google Scholar
  7. Hammerling, M. J., Fritz, B. R., Yoesep, D. J., Carlson, E. D. & Jewett, M. C. In vitro ribosome synthesis and evolution through ribosome display. Nat. Commun. 11, 1108 (2020).
    Article CAS PubMed PubMed Central Google Scholar
  8. Maini, R. et al. Protein synthesis with ribosomes selected for the incorporation of β-amino acids. Biochemistry 54, 3694–3706 (2015).
    Article CAS PubMed Google Scholar
  9. Carlson, E. D. et al. Engineered ribosomes with tethered subunits for expanding biological function. Nat. Commun. 10, 3920 (2019).
    Article PubMed PubMed Central Google Scholar
  10. Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).
    Article CAS PubMed PubMed Central Google Scholar
  11. Sailer, Z. R. & Harms, M. J. Molecular ensembles make evolution unpredictable. Proc. Natl Acad. Sci. USA 114, 11938–11943 (2017).
    Article CAS PubMed PubMed Central Google Scholar
  12. Sarkisyan, K. S. et al. Local fitness landscape of the green fluorescent protein. Nature 533, 397–401 (2016).
    Article CAS PubMed PubMed Central Google Scholar
  13. Ramakrishnan, V. Ribosome structure and the mechanism of translation. Cell 108, 557–572 (2002).
    Article CAS PubMed Google Scholar
  14. Schmied, W. H. et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444–448 (2018).
    Article CAS PubMed PubMed Central Google Scholar
  15. Fried, S. D., Schmied, W. H., Uttamapinant, C. & Chin, J. W. Ribosome subunit stapling for orthogonal translation in E. coli. Angew. Chem. 127, 12982–12985 (2015).
    Article PubMed Google Scholar
  16. Liu, C. C., Jewett, M. C., Chin, J. W. & Voigt, C. A. Toward an orthogonal central dogma. Nat. Chem. Biol. 14, 103–106 (2018).
    Article PubMed PubMed Central Google Scholar
  17. Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. & Chin, J. W. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441–444 (2010).
    Article CAS PubMed Google Scholar
  18. Orelle, C. et al. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119–124 (2015).
    Article CAS PubMed Google Scholar
  19. Rackham, O. & Chin, J. W. A network of orthogonal ribosome· mRNA pairs. Nat. Chem. Biol. 1, 159–166 (2005).
    Article CAS PubMed Google Scholar
  20. Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat. Biotechnol. 25, 770–777 (2007).
    Article PubMed Google Scholar
  21. Liu, Y., Kim, D. S. & Jewett, M. C. Repurposing ribosomes for synthetic biology. Curr. Opin. Chem. Biol. 40, 87–94 (2017).
    Article CAS PubMed PubMed Central Google Scholar
  22. Liu, F., Bratulić, S., Costello, A., Miettinen, T. P. & Badran, A. H. Directed evolution of rRNA improves translation kinetics and recombinant protein yield. Nat. Commun. 12, 5638 (2021).
    Article CAS PubMed PubMed Central Google Scholar
  23. Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).
    Article CAS PubMed Google Scholar
  24. Melo Czekster, C., Robertson, W. E., Walker, A. S., Söll, D. & Schepartz, A. In vivo biosynthesis of a β-amino acid-containing protein. J. Am. Chem. Soc. 138, 5194–5197 (2016).
    Article CAS PubMed Google Scholar
  25. Chin, J. W. Expanding and reprogramming the genetic code. Nature 550, 53–60 (2017).
    Article CAS PubMed Google Scholar
  26. Jewett, M. C., Fritz, B. R., Timmerman, L. E. & Church, G. M. In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation. Mol. Syst. Biol. 9, 678 (2013).
    Article CAS PubMed PubMed Central Google Scholar
  27. Hui, A. & de Boer, H. A. Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli. Proc. Natl Acad. Sci. USA 84, 4762–4766 (1987).
    Article CAS PubMed PubMed Central Google Scholar
  28. Rackham, O. & Chin, J. W. Cellular logic with orthogonal ribosomes. J. Am. Chem. Soc. 127, 17584–17585 (2005).
    Article CAS PubMed Google Scholar
  29. Cho, N. et al. De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries. Nat. Commun. 6, 8351 (2015).
    Article CAS PubMed Google Scholar
  30. Yoo, J. I., Daugherty, P. S. & O’Malley, M. A. Bridging non-overlapping reads illuminates high-order epistasis between distal protein sites in a GPCR. Nat. Commun. 11, 690 (2020).
    Article CAS PubMed PubMed Central Google Scholar
  31. Borgström, E. et al. Phasing of single DNA molecules by massively parallel barcoding. Nat. Commun. 6, 7173 (2015).
    Article PubMed Google Scholar
  32. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
    Article CAS PubMed Google Scholar
  33. Asai, T., Zaporojets, D., Squires, C. & Squires, C. L. An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria. Proc. Natl Acad. Sci. USA 96, 1971–1976 (1999).
    Article CAS PubMed PubMed Central Google Scholar
  34. Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).
    Article PubMed PubMed Central Google Scholar
  35. Watkins, A. M., Rangan, R. & Das, R. FARFAR2: improved de novo Rosetta prediction of complex global RNA folds. Structure 28, 963–976 (2020).
  36. Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Mol. Biol. 22, 336–341 (2015).
    Article CAS PubMed PubMed Central Google Scholar
  37. Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat. Methods 14, 75–82 (2017).
    Article CAS PubMed Google Scholar
  38. Aleksashin, N. A. et al. Assembly and functionality of the ribosome with tethered subunits. Nat. Commun. 10, 930 (2019).
    Article PubMed PubMed Central Google Scholar
  39. Lee, J., Schwarz, K. J., Kim, D. S., Moore, J. S. & Jewett, M. C. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro. Nat. Commun. 11, 4304 (2020).
    Article CAS PubMed PubMed Central Google Scholar
  40. Lee, J. et al. Expanding the limits of the second genetic code with ribozymes. Nat. Commun. 10, 5097 (2019).
    Article PubMed PubMed Central Google Scholar
  41. Lee, J., Torres, R., Byrom, M., Ellington, A. D. & Jewett, M. C. Ribosomal incorporation of cyclic β-amino acids into peptides using in vitro translation. Chem. Commun. 56, 5597–5600 (2020).
    Article CAS Google Scholar
  42. Kappel, K. et al. De novo computational RNA modeling into cryo-EM maps of large ribonucleoprotein complexes. Nat. Methods 15, 947–954 (2018).
    Article CAS PubMed PubMed Central Google Scholar
  43. Sun, Q., Vila-Sanjurjo, A. & O’Connor, M. Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit–subunit interactions on the 70S ribosome. Nucleic Acids Res. 39, 3321–3330 (2011).
    Article CAS PubMed Google Scholar
  44. Zhang, L. et al. The structural basis for inhibition of ribosomal translocation by viomycin. Proc. Natl Acad. Sci. USA 117, 10271–10277 (2020).
    Article CAS PubMed PubMed Central Google Scholar
  45. Pulk, A., Maiväli, Ü. & Remme, J. Identification of nucleotides in E. coli 16S rRNA essential for ribosome subunit association. RNA 12, 790–796 (2006).
    Article CAS PubMed PubMed Central Google Scholar
  46. Aleksashin, N. A. et al. A fully orthogonal system for protein synthesis in bacterial cells. Nat. Commun. 11, 1858 (2020).
    Article CAS PubMed PubMed Central Google Scholar
  47. Masella, A. P., Bartram, A. K., Truszkowski, J. M., Brown, D. G. & Neufeld, J. D. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinf. 13, 31 (2012).
    Article CAS Google Scholar
  48. Incarnato, D., Morandi, E., Simon, L. M. & Oliviero, S. RNA Framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications. Nucleic Acids Res. 46, e97–e97 (2018).
    Article PubMed PubMed Central Google Scholar
  49. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
    Article CAS PubMed Google Scholar
  50. Pintilie, G. D., Zhang, J., Goddard, T. D., Chiu, W. & Gossard, D. C. Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J. Struct. Biol. 170, 427–438 (2010).
    Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This work was supported by the National Science Foundation (grant no. MCB-1716766), the Human Frontiers Science Program (grant no. RGP0015/2017), the Army Research Office (grant no. W911NF-16-1-0372), all to M.C.J. R.D. thanks the NIGMS MIRA R35 award for funding. We thank J. Lucks and M. Evans at Northwestern and R. Kretsch at Stanford for discussions. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (grant no. U24 GM129541). The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the US Government or the National Institutes of Health.

Author information

Author notes

  1. Do Soon Kim
    Present address: Inceptive Nucleics, Inc., Palo Alto, CA, USA
  2. Andrew Watkins
    Present address: Prescient Design, Genentech, South San Francisco, CA, USA
  3. Erik Bidstrup
    Present address: Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
  4. Joongoo Lee
    Present address: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea
  5. These authors contributed equally: Do Soon Kim, Andrew Watkins.

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA
    Do Soon Kim, Erik Bidstrup, Joongoo Lee, Camila Kofman, Emily Roney & Michael C. Jewett
  2. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA
    Do Soon Kim, Erik Bidstrup, Joongoo Lee, Camila Kofman, Emily Roney & Michael C. Jewett
  3. Department of Biochemistry, Stanford University, Stanford, CA, USA
    Andrew Watkins, Ved Topkar & Rhiju Das
  4. Department of Chemistry, University of Illinois Urbana Champaign, Champaign, IL, USA
    Kevin J. Schwarz
  5. Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA, USA
    Yan Liu
  6. Department of Bioengineering, Stanford University, Stanford, CA, USA
    Grigore Pintilie
  7. Department of Physics, Stanford University, Stanford, CA, USA
    Rhiju Das

Authors

  1. Do Soon Kim
  2. Andrew Watkins
  3. Erik Bidstrup
  4. Joongoo Lee
  5. Ved Topkar
  6. Camila Kofman
  7. Kevin J. Schwarz
  8. Yan Liu
  9. Grigore Pintilie
  10. Emily Roney
  11. Rhiju Das
  12. Michael C. Jewett

Contributions

D.S.K., M.C.J., A.W. and R.D. conceived the study and designed experiments. D.S.K., E.B., E.R. and C.K. worked on establishing the Evolink method. D.S.K. and E.B. carried out experiments for tethered ribosome evolution, orthogonal GFP expression and cellular growth rates. J.L. and K.J.S. carried out experiments for noncanonical amino acid incorporation. D.S.K. and A.W. analyzed results and designed the libraries. A.W. carried out computational modeling. V.T. performed chemical mapping experiments. D.S.K., V.T. and Y.L. performed cryo-EM sample preparation and data collection. V.T. and G.P. performed cryo-EM data analysis and structure determination. D.S.K., A.W., M.C.J. and R.D. wrote the manuscript with participation by all authors.

Corresponding author

Correspondence toMichael C. Jewett.

Ethics declarations

Competing interests

M.C.J. and D.S.K. are coinventors on the US provisional patent application that incorporates discoveries described in this manuscript. M.C.J. has a financial interest in Pearl Bio, and his interests are reviewed and managed by Northwestern University in accordance with their competing interest policies. All other authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Luc Jaeger, Jérôme Waldispühl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

a, A clonal sample of the tethered ribosome (Ribo-T v2) is linearized using different oligos compatible with multiple ligation protocols. b, From the different ligation products, generation of final amplicon for next-generation sequencing can happen with a wide range of ligation methods and starting template amounts in the PCR. Gel data representative of two independent experiments.

Source data

a–c, Positively enriched genotypes (purple) and negative enriched genotypes (dark gray) can be tracked throughout multiple time points throughout selection. Genotypes that drop out during selection can also be identified (light gray). Corresponding heat maps that reveal trends in selected tether lengths also helped inform designs. Generally, across the three libraries tested in this work, (a) the Broad Sampling Library, (b) the Designed Junction Library, and (c) the Designed Junction + Length Refined Library, log2-fold enrichment values between -6 to 6 are observed. Enrichment and heatmap data representative of three independent experiments.

Extended Data Fig. 3 Score vs. Root-Mean-Standard-Deviation analysis of FARFAR2 simulations of enriched tether sequences.

a–d, For the Broad Sampling Library, we observe striking differences between simulations that constrained (blue) or did not constrain (orange) 3D structures of the Tether-H101 junction. Of the four modeled genotypes, two sequence (c,d) exhibit particularly substantial differences, hinting at structural instability in the Tether-H101 junction. e–h, When similar simulations are performed with enriched tether sequences from the Designed Junction Library (designed sequences at the Tether-H101 junction), the results of FARFAR2 simulations reach similarly low scores in constrained vs. unconstrained modeling runs.

Extended Data Fig. 4 Representative constrained and unconstrained 3D models of Designed Junction Library winner.

The winning genotype from Fig. 4h was modeled using Rosetta, and representative outputs are shown. In both the (a) unconstrained and (b) constrained model, the Designed Junction residues are predicted to base pair, reinforcing structural stability to this region.

Extended Data Fig. 5 Chemical reactivity of tethers to DMS in whole polysomes.

Targeted structure probing was performed on the tethers of both RiboTv2 and RiboTv3 polysomes via DMS-MaPseq. The per-nucleotide chemical reactivities of the tethers and their adjacent rRNA stems can be seen in the figure for both RiboTv2 (a) and RiboTv3 (b). Gray shaded nucleotides represent U and G residues that are not modified by DMS.

Extended Data Fig. 6 Raw Cryo-EM micrographs of Ribo-Tv3 polysomes.

Single-particle Cryo-EM was carried out on RiboTv3 polysomes. a, A representative raw micrograph shows that RiboTv3 polysomes look like characteristic ‘beads on a string’ as expected for actively translating ribosomes. b, A tethered ribosome that has dissociated from an mRNA looks like an open clamshell, as would be expected for a tethered ribosome. The large and small subunits are indicated by white arrows.

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Kim, D.S., Watkins, A., Bidstrup, E. et al. Three-dimensional structure-guided evolution of a ribosome with tethered subunits.Nat Chem Biol 18, 990–998 (2022). https://doi.org/10.1038/s41589-022-01064-w

Download citation