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Structural basis for DNA targeting by the Tn7 transposon

Nature Structural & Molecular Biology volume 29, pages 143–151 (2022)Cite this article

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Abstract

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

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Fig. 1: TnsC forms oligomers in a DNA- and AMPPnP-dependent manner.
Fig. 2: TnsC-specific insertions decorate the surface of TnsCS-A225V.
Fig. 3: Cryo-EM structure of TnsCS-A225V bound to the 20-7-20 DNA.
Fig. 4: Asp402 orients the C-terminal region of TnsC.
Fig. 5: TnsC binds DNA through the ISM.
Fig. 6: The N-terminal side of the TnsC ring faces the DNA distortion.
Fig. 7: Model for DNA targeting by the Tn7 element.

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Data availability

Coordinates and structure factors for the TnsCS-A225V crystal structure are available in the RCSB Protein Data Bank (PDB 7MBW). The cryo-EM map for the structure of TnsCS-A225V bound to DNA (class 2.1.B) has been deposited in the EMDB (EMD-23757) and the associated model has been deposited in the RCSB Protein Data Bank (PDB 7MCS).

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Acknowledgements

We thank N. Craig for critical reading of the manuscript, J. Rubinstein (SickKids, Toronto) for access to his Tecnai F20 during the early stages of this project and FEMR personnel at McGill University for data collection assistance. This work was funded by the Canadian Institutes of Health Research (PJT-155941, A.G.) and the National Institutes of Health (R01GM129118, J.E.P.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry, McGill University, Montreal, Quebec, Canada

    Yao Shen, Josue Gomez-Blanco & Alba Guarné

  2. Centre de Recherche and Biologie Structurale, McGill University, Montreal, Quebec, Canada

    Yao Shen, Josue Gomez-Blanco, Joaquin Ortega & Alba Guarné

  3. Department of Microbiology, Cornell University, Ithaca, NY, USA

    Michael T. Petassi & Joseph E. Peters

  4. Department of Anatomy and Cell Biology, McGill University Montreal, Montreal, Quebec, Canada

    Joaquin Ortega

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Contributions

Y.S. and A.G. conceived the study and interpreted the data. Y.S. characterized the TnsC complexes, collected data, and determined and analyzed the crystal and cryo-EM structures. J.G.-B. and J.O. assisted with cryo-EM data collection and processing. M.T.P. and J.E.P. performed and interpreted the lambda hop assays. A.G. and J.E.P. obtained funding for this study. A.G. prepared the manuscript, with input from all authors.

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Correspondence to Alba Guarné.

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Competing interests

Cornell University has filed patent applications with J.E.P. as inventor involving CRISPR-Cas systems associated with transposons that are not related to this work. The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Video 1

Conformational rearrangements of the C-terminal region of TnsCS-A225V. In the conformation seen in the crystal structure (first frame), TnsCS-A225V would not be able to form a ring because the C-terminal region of TnsC (residues 381–487, colored salmon) would overlap with the oligomerization interface. Therefore, to assemble the ring, this region must swivel ~180° around residue Asp402 to orient the C-terminal region outwards of the ring (last frame). The N-terminal extension (TnsD-interaction region) preceding the AAA+ domain and INS1 are colored in blue and teal for reference.

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Shen, Y., Gomez-Blanco, J., Petassi, M.T. et al. Structural basis for DNA targeting by the Tn7 transposon. Nat Struct Mol Biol 29, 143–151 (2022). https://doi.org/10.1038/s41594-022-00724-8

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