Yan Liming, Yang Yunxiang, Li Mingyu, Zhang Ying, Zheng Litao, Ge Ji, Huang Yucen C, Liu Zhenyu, Wang Tao, Gao Shan, Zhang Ran, Huang Yuanyun Y, Guddat Luke W, Gao Yan, Rao Zihe, Lou Zhiyong
MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
Cell. 2021 Jun 24;184(13):3474-3485.e11. doi: 10.1016/j.cell.2021.05.033. Epub 2021 May 24.
The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (GpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.
mRNA加帽和校对在严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的复制和转录过程中起着至关重要的作用。在此，我们展示了SARS-CoV-2复制转录复合体（RTC）的冷冻电镜结构，其形式被确定为Cap(0)-RTC，它结合了一个共转录加帽复合体（CCC），该复合体由nsp12 NiRAN、nsp9、具有N端外切核糖核酸酶（ExoN）和C端N7-甲基转移酶（N7-MTase）的双功能nsp14以及作为nsp14辅因子的nsp10组成。Nsp9和nsp12 NiRAN将nsp10/nsp14招募到Cap(0)-RTC中，形成N7-CCC，从而在mRNA前体的5'端产生帽(0)（GpppA）。冷冻电镜观察到的Cap(0)-RTC二聚体形式表明，nsp14 ExoN存在一种反式回溯机制，以便与聚合酶nsp12协同促进RNA的校对。这些结果不仅为理解SARS-CoV-2 mRNA的共转录修饰提供了结构基础，也揭示了SARS-CoV-2如何维持复制保真度。
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