小柯机器人

日本东京大学Osamu Nureki研究小组揭示转座子相关TnpB酶的冷冻电镜结构。相关论文于2023年4月5日在线发表在《自然》杂志上。

研究人员报告了Deinococcus radiodurans ISDra2 TnpB与其同源的ωRNA和目标DNA复合物的冷冻电镜（cryo-EM）结构。在该结构中，ωRNA采用了一个意想不到的结构，形成了一个假结，这在Cas12酶的所有向导RNA中是保守的。此外，该结构以及功能分析揭示了紧凑的TnpB如何识别ωRNA并切割与向导互补的目标DNA。TnpB与Cas12酶的结构比较表明，CRISPR-Cas12效应器通过不对称的二聚体形成或不同的REC2插入，获得了识别向导RNA-靶DNA异源二聚体的前间隔序列邻近基序末端的能力，使得参与CRISPR-Cas适应性免疫。总之，这项发现为TnpB的功能提供了机理上的见解，并推进了人们对从转座子编码的TnpB蛋白到CRISPR-Cas12效应物演化的理解。

据了解，2类V型CRISPR效应器Cas12被认为是从IS200/IS605超家族的转座子相关TnpB蛋白演化而来。最近的研究已经确定TnpB蛋白是微型的RNA引导的DNA内切酶。TnpB与单一的长RNA（ωRNA）结合，并切割与ωRNA引导互补的双链DNA目标。然而，TnpB的RNA引导的DNA裂解机制以及它与Cas12酶的进化关系仍然是未知的。

附：英文原文

Title: Cryo-EM structure of the transposon-associated TnpB enzyme

Author: 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

Issue&Volume: 2023-04-05

Abstract: The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. 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.

DOI: 10.1038/s41586-023-05933-9

Source: https://www.nature.com/articles/s41586-023-05933-9

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
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