Cryo-EM structure of the transposon-associated TnpB enzyme.
Cryo-EM structure of the transposon-associated TnpB enzyme.
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DOI：
10.1038/s41586-023-05933-9
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发表时间：
2023-04
期刊：
影响因子：
64.8
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
--
作者：
Nakagawa, Ryoya;Hirano, Hisato;Omura, Satoshi N.;Nety, Suchita;Kannan, Soumya;Altae-Tran, Han;Yao, Xiao;Sakaguchi, Yuriko;Ohira, Takayuki;Wu, Wen Y.;Nakayama, Hiroshi;Shuto, Yutaro;Tanaka, Tatsuki;Sano, Fumiya K.;Kusakizako, Tsukasa;Kise, Yoshiaki;Itoh, Yuzuru;Dohmae, Naoshi;van der Oost, John;Suzuki, Tsutomu;Zhang, Feng;Nureki, Osamu
2类V型CRISPR效应子Cas 12被认为是从转座子相关TnpB蛋白的IS 200/IS 605超家族进化而来的。最近的研究已经确定TnpB蛋白作为微型RNA引导的DNA内切酶。TnpB与单个长RNA（ωRNA）结合并切割与ωRNA向导互补的双链DNA靶标。然而，TnpB的RNA引导的DNA切割机制及其与Cas 12酶的进化关系仍然未知。本文报道了耐辐射球菌ISDra 2 TnpB与其同源ωRNA和靶DNA复合物的冷冻电镜（cryo-EM）结构。在结构中，ωRNA采用了意想不到的结构并形成了假结，这在Cas 12酶的所有指导RNA中是保守的。此外，结构，沿着我们的功能分析，揭示了紧凑TnpB如何识别ωRNA并切割与指导互补的靶DNA。TnpB与Cas 12酶的结构比较表明，CRISPR-Cas 12效应子通过不对称二聚体形成或不同的REC 2插入获得了识别引导RNA-靶DNA异源双链体的原型间隔区相邻基序远端的能力，从而能够参与CRISPR-Cas适应性免疫。总的来说，我们的研究结果为TnpB功能提供了机制性见解，并推进了我们对从转座子编码的TnpB蛋白到CRISPR-Cas 12效应子进化的理解。对耐辐射球菌ISDra 2 TnpB与其同源ωRNA和靶DNA复合物的冷冻电子显微镜分析提供了对TnpB功能机制和CRISPR-Cas 12效应子进化的见解。
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases. 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. Cryo-electron microscopy analysis of the Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA provides insights into the mechanism of TnpB function and the evolution of CRISPR–Cas12 effectors.
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DOI：
10.1126/science.abb1400
发表时间：
2020-07-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Pausch P;Al-Shayeb B;Bisom-Rapp E;Tsuchida CA;Li Z;Cress BF;Knott GJ;Jacobsen SE;Banfield JF;Doudna JA
通讯作者：
Doudna JA
DOI：
10.1126/science.aav4294
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Harrington LB;Burstein D;Chen JS;Paez-Espino D;Ma E;Witte IP;Cofsky JC;Kyrpides NC;Banfield JF;Doudna JA
通讯作者：
Doudna JA
影响因子：
48
作者：
Barad BA;Echols N;Wang RY;Cheng Y;DiMaio F;Adams PD;Fraser JS
通讯作者：
Fraser JS
影响因子：
64.8
作者：
Hirano, Seiichi;Kappel, Kalli;Altae-Tran, Han;Faure, Guilhem;Wilkinson, Max E.;Kannan, Soumya;Demircioglu, F. Esra;Yan, Rui;Shiozaki, Momoko;Yu, Zhiheng;Makarova, Kira S.;Koonin, Eugene, V;Macrae, Rhiannon K.;Zhang, Feng
通讯作者：
Zhang, Feng
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D