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Fusion RNAs in Crystallographic Studies of Double-Stranded RNA from Trypanosome RNA Editing

  • Blaine H. M. MooersEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1240)

Abstract

Head-to-head fusions of two identical double-stranded fragments of RNA can be designed to self-assemble from a single RNA species and form a double-stranded helix with a twofold rotation axis relating the two strands. These symmetrical RNA molecules are more likely to crystallize without end-on-end statistical packing disorder because the two halves of the molecule are identical. This approach can be used to study many fragments of double-stranded RNA or many isolated helical domains from large single-stranded RNAs that may not yet be amenable to high-resolution studies by crystallography or NMR. We used fusion RNAs to study one (the U-helix) of three functional domains formed when guide RNA binds to its cognate pre-edited mRNA from the trypanosome RNA editing system. The U-helix forms when the 3′ oligo(U) tail of the guide RNA (gRNA) binds to the purine-rich, pre-edited mRNA upstream from the current RNA editing site. Fusion RNAs 16-and 32-base pairs in length formed crystals that gave diffraction to 1.37 and 1.05 Å respectively. We provide the composition of a fusion RNA crystallization screen and describe the X-ray data collection, structure determination, and refinement of the crystal structures of fusion RNAs.

Key words

RNA–RNA interactions Fusion RNA RNA self-assembly RNA dimerization RNA molecular symmetry trans-Acting RNA RNA crystallization screen RNA crystallography RNA design RNA engineering RNA structure Polyuridylation Poly(U) Oligo(U) 3′ Tail U-tail U-helix G · U wobble base pairs X-ray diffraction data collection Crystal cryoprotection 

Notes

Acknowledgements

This research was supported by grants from the Presbyterian Health Foundation (PHF #1545-Mooers), Oklahoma Center for the Advancement of Science and Technology (HR08-138), and the NIH-NIAID (R01-AI088011). We thank Dr. Tzanko Doukov for help with data collection at Stanford Synchrotron Radiation Lightsource (SSRL) beam line 7–1. The Structural Molecular Biology Program at SSRL is supported by DOE-OBER, NIH-NCRR (P41RR001209), and NIH-NIGMS. This work was also supported in part by an Institutional Development Award (IDeA) from the NIH-GMS under grant P20-GM103640.

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Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA

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