دورية أكاديمية
Cryo-EM structure of the transposon-associated TnpB enzyme.
| العنوان: | Cryo-EM structure of the transposon-associated TnpB enzyme. |
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| المؤلفون: | Nakagawa R; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Hirano H; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Omura SN; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Nety S; Broad Institute of MIT and Harvard, Cambridge, MA, USA.; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA.; Howard Hughes Medical Institute, Cambridge, MA, USA., Kannan S; Broad Institute of MIT and Harvard, Cambridge, MA, USA.; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA.; Howard Hughes Medical Institute, Cambridge, MA, USA., Altae-Tran H; Broad Institute of MIT and Harvard, Cambridge, MA, USA.; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA.; Howard Hughes Medical Institute, Cambridge, MA, USA., Yao X; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan., Sakaguchi Y; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan., Ohira T; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan., Wu WY; Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands., Nakayama H; Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Saitama, Japan., Shuto Y; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Tanaka T; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Sano FK; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Kusakizako T; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Kise Y; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.; Curreio, The University of Tokyo, Tokyo, Japan., Itoh Y; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan., Dohmae N; Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Saitama, Japan., van der Oost J; Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands., Suzuki T; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan., Zhang F; Broad Institute of MIT and Harvard, Cambridge, MA, USA.; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA.; Howard Hughes Medical Institute, Cambridge, MA, USA., Nureki O; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan. nureki@bs.s.u-tokyo.ac.jp.; Curreio, The University of Tokyo, Tokyo, Japan. nureki@bs.s.u-tokyo.ac.jp. |
| المصدر: | Nature [Nature] 2023 Apr; Vol. 616 (7956), pp. 390-397. Date of Electronic Publication: 2023 Apr 05. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't; Research Support, N.I.H., Extramural |
| اللغة: | English |
| بيانات الدورية: | Publisher: Nature Publishing Group Country of Publication: England NLM ID: 0410462 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1476-4687 (Electronic) Linking ISSN: 00280836 NLM ISO Abbreviation: Nature Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Basingstoke : Nature Publishing Group Original Publication: London, Macmillan Journals ltd. |
| مواضيع طبية MeSH: | Bacterial Proteins*/chemistry , Bacterial Proteins*/metabolism , Bacterial Proteins*/ultrastructure , Cryoelectron Microscopy* , DNA Transposable Elements*/genetics , Endodeoxyribonucleases*/chemistry , Endodeoxyribonucleases*/metabolism , Endodeoxyribonucleases*/ultrastructure , Deinococcus*/enzymology , Deinococcus*/genetics, CRISPR-Associated Proteins/chemistry ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; DNA/ultrastructure ; RNA, Guide, CRISPR-Cas Systems/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Guide, CRISPR-Cas Systems/ultrastructure ; Substrate Specificity |
| مستخلص: | The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins 1 . Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases 2,3 . TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors. (© 2023. The Author(s).) |
| References: | Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020). (PMID: 10.1038/s41579-019-0299-x31857715) Altae-Tran, H. et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science 374, 57–65 (2021). (PMID: 10.1126/science.abj6856345916438929163) Karvelis, T. et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature 599, 692–696 (2021). (PMID: 10.1038/s41586-021-04058-1346197448612924) Hille, F. et al. The biology of CRISPR–Cas: backward and forward. Cell 172, 1239–1259 (2018). (PMID: 10.1016/j.cell.2017.11.03229522745) Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, 2579–2586 (2012). (PMID: 10.1073/pnas.1208507109) Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). (PMID: 10.1126/science.1225829227452496286148) Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015). (PMID: 10.1016/j.cell.2015.09.038264222274638220) Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR–Cas systems. Mol. Cell 60, 385–397 (2015). (PMID: 10.1016/j.molcel.2015.10.008265937194660269) Yan, W. X. et al. Functionally diverse type V CRISPR–Cas systems. Science 363, 88–91 (2019). (PMID: 10.1126/science.aav727130523077) Burstein, D. et al. New CRISPR–Cas systems from uncultivated microbes. Nature 542, 237–241 (2017). (PMID: 10.1038/nature2105928005056) Harrington, L. B. et al. Programmed DNA destruction by miniature CRISPR–Cas14 enzymes. Science 362, 839 (2018). (PMID: 10.1126/science.aav4294303374556659742) Pausch, P. et al. Crispr–CasФ from huge phages is a hypercompact genome editor. Science 69, 333–337 (2020). (PMID: 10.1126/science.abb1400) Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019). (PMID: 10.1126/science.aax9181311717066659118) Urbaitis, T. et al. A new family of CRISPR‐type V nucleases with C‐rich PAM recognition. EMBO Rep. 23, e55481 (2022). (PMID: 10.15252/embr.202255481362685819724661) Wu, W. Y. et al. The miniature CRISPR–Cas12m effector binds DNA to block transcription. Mol. Cell 82, 4487–4502.e7 (2022). (PMID: 10.1016/j.molcel.2022.11.00336427491) Schuler, G., Hu, C. & Ke, A. Structural basis for RNA-guided DNA cleavage by IscB–ωRNA and mechanistic comparison with Cas9. Science 376, 1476–1481 (2022). (PMID: 10.1126/science.abq72203561737110041819) Kato, K. et al. Structure of the IscB–ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR–Cas9. Nat. Commun. 13, 6719 (2022). (PMID: 10.1038/s41467-022-34378-3363445049640706) Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014). (PMID: 10.1038/nature13579250793184176945) Takeda, S. N. et al. Structure of the miniature type V-F CRISPR–Cas effector enzyme. Mol. Cell 81, 558–570.e3 (2021). (PMID: 10.1016/j.molcel.2020.11.03533333018) Swarts, D. C. & Jinek, M. Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a. Mol. Cell 73, 589–600.e4 (2019). (PMID: 10.1016/j.molcel.2018.11.021306392406858279) Liu, J. J. et al. CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566, 218–223 (2019). (PMID: 10.1038/s41586-019-0908-x307187746662743) Gomes-Filho, J. V. et al. Sense overlapping transcripts in IS1341-type transposase genes are functional non-coding RNAs in archaea. RNA Biol. 12, 490–500 (2015). (PMID: 10.1080/15476286.2015.1019998258064054615843) Kim, D. Y. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat. Biotechnol. 40, 94–102 (2022). (PMID: 10.1038/s41587-021-01009-z34475560) Wang, Y. et al. Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease. Cell Rep. 40, 111481 (2022). (PMID: 10.1016/j.celrep.2022.111418) Schmid-Burgk, J. L. et al. Highly parallel profiling of Cas9 variant specificity. Mol. Cell 78, 794–800.e8 (2020). (PMID: 10.1016/j.molcel.2020.02.023321875297370240) Yang, H., Gao, P., Rajashankar, K. R. & Patel, D. J. PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR–Cas endonuclease. Cell 167, 1814–1828.e12 (2016). (PMID: 10.1016/j.cell.2016.11.053279847295278635) Hirano, S. et al. Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA. Nature 610, 575–581 (2022). (PMID: 10.1038/s41586-022-05324-6362243869581776) 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). (PMID: 10.1038/nmeth.416928165473) Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020). (PMID: 10.1038/s41592-020-00990-833257830) Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003). (PMID: 10.1016/j.jmb.2003.07.01314568533) Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). (PMID: 10.1038/s41586-021-03819-2342658448371605) Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D D60, 2126–2132 (2004). (PMID: 10.1107/S0907444904019158) Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018). (PMID: 10.1107/S2059798318006551) Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010). (PMID: 10.1107/S090744490904207320057044) Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015). (PMID: 10.1038/nmeth.3541262803284589481) Zi Tan, Y. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017). (PMID: 10.1038/nmeth.4347) Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). (PMID: 10.1002/pro.394332881101) Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019). (PMID: 10.1038/s41587-019-0032-3308090266533916) Söding, J. Protein homology detection by HMM–HMM comparison. Bioinformatics 21, 951–960 (2005). (PMID: 10.1093/bioinformatics/bti12515531603) |
| معلومات مُعتمدة: | R01 HG009761 United States HG NHGRI NIH HHS; T32 GM007753 United States GM NIGMS NIH HHS; T32 GM144273 United States GM NIGMS NIH HHS |
| المشرفين على المادة: | 0 (Bacterial Proteins) 0 (CRISPR-Associated Proteins) 9007-49-2 (DNA) 0 (DNA Transposable Elements) 0 (RNA, Guide, CRISPR-Cas Systems) EC 3.1.- (Endodeoxyribonucleases) |
| تواريخ الأحداث: | Date Created: 20230405 Date Completed: 20230419 Latest Revision: 20240328 |
| رمز التحديث: | 20240329 |
| مُعرف محوري في PubMed: | PMC10097598 |
| DOI: | 10.1038/s41586-023-05933-9 |
| PMID: | 37020030 |
| قاعدة البيانات: | MEDLINE |
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Full Text Finder | تدمد: | 1476-4687 |
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| DOI: | 10.1038/s41586-023-05933-9 |