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Title: Hatchet ribozyme structure and implications for cleavage mechanism

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.

Authors:
 [1];  [2];  [1];  [2]; ORCiD logo [3];  [4];  [5];  [2]; ORCiD logo [1]
  1. Zhejiang Univ., Hangzhou (China). Life Science Inst.
  2. Leopold Franzens Univ., Innsbruck (Austria). Inst. of Organic Chemistry
  3. Ecole Polytechnique Federale Lausanne (Switzlerland)
  4. Memorial Sloan-Kettering Cancer Center, New York, NY (United States)
  5. Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Southern Univ. of Science and Technology, Shenzhen (China)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE; National Institutes of Health (NIH)
OSTI Identifier:
1531009
Grant/Contract Number:  
[NIH-P30CA008748; NIH-1U19CA179564,]
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
[ Journal Volume: 116; Journal Issue: 22]; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; hatchet; ribozyme; cleavage; catalysis; noncoding RNA

Citation Formats

Zheng, Luqian, Falschlunger, Christoph, Huang, Kaiyi, Mairhofer, Elisabeth, Yuan, Shuguang, Wang, Juncheng, Patel, Dinshaw J., Micura, Ronald, and Ren, Aiming. Hatchet ribozyme structure and implications for cleavage mechanism. United States: N. p., 2019. Web. doi:10.1073/pnas.1902413116.
Zheng, Luqian, Falschlunger, Christoph, Huang, Kaiyi, Mairhofer, Elisabeth, Yuan, Shuguang, Wang, Juncheng, Patel, Dinshaw J., Micura, Ronald, & Ren, Aiming. Hatchet ribozyme structure and implications for cleavage mechanism. United States. doi:10.1073/pnas.1902413116.
Zheng, Luqian, Falschlunger, Christoph, Huang, Kaiyi, Mairhofer, Elisabeth, Yuan, Shuguang, Wang, Juncheng, Patel, Dinshaw J., Micura, Ronald, and Ren, Aiming. Tue . "Hatchet ribozyme structure and implications for cleavage mechanism". United States. doi:10.1073/pnas.1902413116.
@article{osti_1531009,
title = {Hatchet ribozyme structure and implications for cleavage mechanism},
author = {Zheng, Luqian and Falschlunger, Christoph and Huang, Kaiyi and Mairhofer, Elisabeth and Yuan, Shuguang and Wang, Juncheng and Patel, Dinshaw J. and Micura, Ronald and Ren, Aiming},
abstractNote = {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.},
doi = {10.1073/pnas.1902413116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = [22],
volume = [116],
place = {United States},
year = {2019},
month = {5}
}

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