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Proc Natl Acad Sci U S A. 2019 May 28;116(22):10783-10791. doi: 10.1073/pnas.1902413116. Epub 2019 May 14.

Hatchet ribozyme structure and implications for cleavage mechanism.

Author information

1
Life Science Institute, Zhejiang University, 310058 Hangzhou, China.
2
Institute of Organic Chemistry, Leopold Franzens University, 6020 Innsbruck, Austria.
3
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
4
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.
5
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; pateld@mskcc.org ronald.micura@uibk.ac.at aimingren@zju.edu.cn.
6
Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China.
7
Institute of Organic Chemistry, Leopold Franzens University, 6020 Innsbruck, Austria; pateld@mskcc.org ronald.micura@uibk.ac.at aimingren@zju.edu.cn.
8
Life Science Institute, Zhejiang University, 310058 Hangzhou, China; pateld@mskcc.org ronald.micura@uibk.ac.at aimingren@zju.edu.cn.

Abstract

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5' nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.

KEYWORDS:

catalysis; cleavage; hatchet; noncoding RNA; ribozyme

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