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-Structure paper
タイトル | Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system. |
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ジャーナル・号・ページ | Nature, Vol. 577, Issue 7789, Page 271-274, Year 2020 |
掲載日 | 2019年12月18日 |
著者 | Tyler S Halpin-Healy / Sanne E Klompe / Samuel H Sternberg / Israel S Fernández / |
PubMed 要旨 | Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically ...Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications. |
リンク | Nature / PubMed:31853065 |
手法 | EM (単粒子) |
解像度 | 2.9 - 3.5 Å |
構造データ | EMDB-20349: VC-Tn6677 multisubunit CRISPR/Cas effector, close conformation. EMDB-20350: VC-Tn6677 multisubunit CRISPR/Cas effector, open conformation. EMDB-20351: VC-Tn6677 multisubunit CRISPR/Cas effector, close conformation. |
由来 |
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キーワード | RNA BINDING PROTEIN/RNA / CRISPR/Cas / Cascade / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex |