Three-dimensional structure-guided evolution of a ribosome with tethered subunits.

Protein Biosynthesis RNA / metabolism Ribosomes / metabolism Synthetic Biology / methods

Journal

Nature chemical biology
ISSN: 1552-4469
Titre abrégé: Nat Chem Biol
Pays: United States
ID NLM: 101231976

Informations de publication

Date de publication:
09 2022
Historique:
received: 22 01 2021
accepted: 17 05 2022
pubmed: 15 7 2022
medline: 25 8 2022
entrez: 14 7 2022
Statut: ppublish

Résumé

RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

Identifiants

DOI: 10.1038/s41589-022-01064-w PMID: 35836020 PMC: PMC9815830
pubmed: 35836020
doi: 10.1038/s41589-022-01064-w
pii: 10.1038/s41589-022-01064-w
pmc: PMC9815830
mid: NIHMS1861865
doi:

Substances chimiques

RNA 63231-63-0

Types de publication

Journal Article Research Support, Non-U.S. Gov't Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.

Langues

eng

Sous-ensembles de citation

IM

Pagination

990-998

Subventions

Organisme : NIGMS NIH HHS
ID : R35 GM122579
Pays : United States
Organisme : NIGMS NIH HHS
ID : U24 GM129541
Pays : United States

Commentaires et corrections

Type : CommentIn

Informations de copyright

© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

Références

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).
pubmed: 12769555 doi: 10.1021/ja035141q
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).
pubmed: 17176075 doi: 10.1021/bi060986a
Dedkova, L. M. et al. β-Puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids. Biochemistry 51, 401–415 (2012).
pubmed: 22145951 doi: 10.1021/bi2016124
Dedkova, L. M. & Hecht, S. M. Expanding the scope of protein synthesis using modified ribosomes. J. Am. Chem. Soc. 141, 6430–6447 (2019).
pubmed: 30901982 pmcid: 6530915 doi: 10.1021/jacs.9b02109
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).
doi: 10.1016/j.cbpa.2015.06.008
Ellefson, J. W. et al. Synthetic evolutionary origin of a proofreading reverse transcriptase. Science 352, 1590–1593 (2016).
pubmed: 27339990 doi: 10.1126/science.aaf5409
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).
pubmed: 32111839 pmcid: 7048773 doi: 10.1038/s41467-020-14705-2
Maini, R. et al. Protein synthesis with ribosomes selected for the incorporation of β-amino acids. Biochemistry 54, 3694–3706 (2015).
pubmed: 25982410 doi: 10.1021/acs.biochem.5b00389
Carlson, E. D. et al. Engineered ribosomes with tethered subunits for expanding biological function. Nat. Commun. 10, 3920 (2019).
pubmed: 31477696 pmcid: 6718428 doi: 10.1038/s41467-019-11427-y
Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).
pubmed: 19935669 pmcid: 2997618 doi: 10.1038/nrm2805
Sailer, Z. R. & Harms, M. J. Molecular ensembles make evolution unpredictable. Proc. Natl Acad. Sci. USA 114, 11938–11943 (2017).
pubmed: 29078365 pmcid: 5691298 doi: 10.1073/pnas.1711927114
Sarkisyan, K. S. et al. Local fitness landscape of the green fluorescent protein. Nature 533, 397–401 (2016).
pubmed: 27193686 pmcid: 4968632 doi: 10.1038/nature17995
Ramakrishnan, V. Ribosome structure and the mechanism of translation. Cell 108, 557–572 (2002).
pubmed: 11909526 doi: 10.1016/S0092-8674(02)00619-0
Schmied, W. H. et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444–448 (2018).
pubmed: 30518861 pmcid: 6525102 doi: 10.1038/s41586-018-0773-z
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).
pubmed: 27570300 doi: 10.1002/ange.201506311
Liu, C. C., Jewett, M. C., Chin, J. W. & Voigt, C. A. Toward an orthogonal central dogma. Nat. Chem. Biol. 14, 103–106 (2018).
pubmed: 29337969 pmcid: 6339509 doi: 10.1038/nchembio.2554
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).
pubmed: 20154731 doi: 10.1038/nature08817
Orelle, C. et al. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119–124 (2015).
pubmed: 26222032 doi: 10.1038/nature14862
Rackham, O. & Chin, J. W. A network of orthogonal ribosome· mRNA pairs. Nat. Chem. Biol. 1, 159–166 (2005).
pubmed: 16408021 doi: 10.1038/nchembio719
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).
pubmed: 17592474 doi: 10.1038/nbt1314
Liu, Y., Kim, D. S. & Jewett, M. C. Repurposing ribosomes for synthetic biology. Curr. Opin. Chem. Biol. 40, 87–94 (2017).
pubmed: 28869851 pmcid: 5696040 doi: 10.1016/j.cbpa.2017.07.012
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).
pubmed: 34561441 pmcid: 8463689 doi: 10.1038/s41467-021-25852-5
Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).
pubmed: 21637198 doi: 10.1038/nprot.2011.331
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).
pubmed: 27086674 doi: 10.1021/jacs.6b01023
Chin, J. W. Expanding and reprogramming the genetic code. Nature 550, 53–60 (2017).
pubmed: 28980641 doi: 10.1038/nature24031
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).
pubmed: 23799452 pmcid: 3964315 doi: 10.1038/msb.2013.31
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).
pubmed: 2440028 pmcid: 305185 doi: 10.1073/pnas.84.14.4762
Rackham, O. & Chin, J. W. Cellular logic with orthogonal ribosomes. J. Am. Chem. Soc. 127, 17584–17585 (2005).
pubmed: 16351070 doi: 10.1021/ja055338d
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).
pubmed: 26387459 doi: 10.1038/ncomms9351
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).
pubmed: 32019920 pmcid: 7000732 doi: 10.1038/s41467-020-14495-7
Borgström, E. et al. Phasing of single DNA molecules by massively parallel barcoding. Nat. Commun. 6, 7173 (2015).
pubmed: 26055759 doi: 10.1038/ncomms8173
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
pubmed: 19363495 doi: 10.1038/nmeth.1318
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).
pubmed: 10051579 pmcid: 26721 doi: 10.1073/pnas.96.5.1971
Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).
pubmed: 22115189 pmcid: 3319429 doi: 10.1186/1748-7188-6-26
Watkins, A. M., Rangan, R. & Das, R. FARFAR2: improved de novo Rosetta prediction of complex global RNA folds. Structure 28, 963–976 (2020).
Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Mol. Biol. 22, 336–341 (2015).
pubmed: 25775265 pmcid: 4429131 doi: 10.1038/nsmb.2994
Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat. Methods 14, 75–82 (2017).
pubmed: 27819661 doi: 10.1038/nmeth.4057
Aleksashin, N. A. et al. Assembly and functionality of the ribosome with tethered subunits. Nat. Commun. 10, 930 (2019).
pubmed: 30804338 pmcid: 6389949 doi: 10.1038/s41467-019-08892-w
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).
pubmed: 32855412 pmcid: 7452890 doi: 10.1038/s41467-020-18001-x
Lee, J. et al. Expanding the limits of the second genetic code with ribozymes. Nat. Commun. 10, 5097 (2019).
pubmed: 31704912 pmcid: 6841967 doi: 10.1038/s41467-019-12916-w
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).
doi: 10.1039/D0CC02121K
Kappel, K. et al. De novo computational RNA modeling into cryo-EM maps of large ribonucleoprotein complexes. Nat. Methods 15, 947–954 (2018).
pubmed: 30377372 pmcid: 6636682 doi: 10.1038/s41592-018-0172-2
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).
pubmed: 21138965 doi: 10.1093/nar/gkq1253
Zhang, L. et al. The structural basis for inhibition of ribosomal translocation by viomycin. Proc. Natl Acad. Sci. USA 117, 10271–10277 (2020).
pubmed: 32341159 pmcid: 7229676 doi: 10.1073/pnas.2002888117
Pulk, A., Maiväli, Ü. & Remme, J. Identification of nucleotides in E. coli 16S rRNA essential for ribosome subunit association. RNA 12, 790–796 (2006).
pubmed: 16556933 pmcid: 1440916 doi: 10.1261/rna.2275906
Aleksashin, N. A. et al. A fully orthogonal system for protein synthesis in bacterial cells. Nat. Commun. 11, 1858 (2020).
pubmed: 32313034 pmcid: 7170887 doi: 10.1038/s41467-020-15756-1
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).
doi: 10.1186/1471-2105-13-31
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).
pubmed: 29893890 pmcid: 6144828 doi: 10.1093/nar/gky486
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).
pubmed: 28165473 doi: 10.1038/nmeth.4169
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).
pubmed: 20338243 pmcid: 2874196 doi: 10.1016/j.jsb.2010.03.007

Auteurs

Do Soon Kim (DS)

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.
Inceptive Nucleics, Inc., Palo Alto, CA, USA.
ORCID: 0000-0003-1682-2829

Andrew Watkins (A)

Department of Biochemistry, Stanford University, Stanford, CA, USA.
Prescient Design, Genentech, South San Francisco, CA, USA.

Erik Bidstrup (E)

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.
Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
ORCID: 0000-0001-9014-2042

Joongoo Lee (J)

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.
Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea.

Ved Topkar (V)

Department of Biochemistry, Stanford University, Stanford, CA, USA.

Camila Kofman (C)

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.

Kevin J Schwarz (KJ)

Department of Chemistry, University of Illinois Urbana Champaign, Champaign, IL, USA.

Yan Liu (Y)

Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA, USA.
ORCID: 0000-0001-5683-8695

Grigore Pintilie (G)

Department of Bioengineering, Stanford University, Stanford, CA, USA.

Emily Roney (E)

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.

Rhiju Das (R)

Department of Biochemistry, Stanford University, Stanford, CA, USA.
Department of Physics, Stanford University, Stanford, CA, USA.
ORCID: 0000-0001-7497-0972

Michael C Jewett (MC)

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA. m-jewett@northwestern.edu.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA. m-jewett@northwestern.edu.
ORCID: 0000-0003-2948-6211

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Classifications MeSH

questionsmedicales.fr › Phénomènes génétiques › Expression des gènes › Biosynthèse des protéines › Three-dimensional structure-guided evolution...
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › Three-dimensional structure-guided evolution...
questionsmedicales.fr › Cellules › Structures cellulaires › Espace intracellulaire › Cytoplasme › Structures cytoplasmiques › Organites › Ribosomes › Three-dimensional structure-guided evolution...
questionsmedicales.fr › Technologie, industrie et agriculture › Ingénierie › Bioingénierie › Biologie synthétique › Three-dimensional structure-guided evolution...