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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
Published on: August 9, 2019
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Structure basis for single-strand nucleic acid targeting by IscB and variants.
Chengtao Xu1, Qi Yang1, Xiaolin Niu1
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, U.S.A.
Biorxiv : the Preprint Server for Biology
|March 18, 2026
Summary
IscB, an ancestor of CRISPR-Cas9, acts as an RNA editor. Structural studies reveal a conformational checkpoint mechanism that controls its nuclease activity, with mutations enhancing RNA-editing efficiency.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- CRISPR-Cas9 technology is derived from the transposon-encoded IscB.
- Engineered IscB and Cas9 serve as RNA editors by modifying their DNA-binding domains.
- Understanding IscB's mechanism is crucial for advancing RNA editing tools.
Purpose of the Study:
- To elucidate the mechanistic underpinnings of IscB's RNA-editing function.
- To determine the structural basis for IscB's target recognition and cleavage.
- To identify strategies for enhancing IscB's RNA-targeting efficiency.
Main Methods:
- Cryo-electron microscopy (cryo-EM) to determine structures of IscB-ssNA complexes.
- Structural analysis to understand conformational changes and nuclease activity.
- Site-directed mutagenesis to probe IscB function and improve efficiency.
Main Results:
- Four cryo-EM structures reveal IscB's interaction with single-stranded nucleic acid (ssNA) targets.
- A conformational checkpoint involving the HNH nuclease domain regulates target cleavage.
- Mutations enhancing ssNA binding or relieving the checkpoint significantly improved RNA-targeting efficiency.
Conclusions:
- IscB employs a conformational checkpoint mechanism for ssNA targeting and cleavage.
- Structural insights guide the engineering of improved IscB-based RNA editors.
- This work provides a foundation for developing more precise and efficient RNA editing technologies.
