Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein
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Strickland, Madeleine, Jonathan Catazaro, Rohith Rajasekaran, et al. “Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein.” Journal of the American Chemical Society 141, no. 4 (2019): 1430–34. https://doi.org/10.1021/jacs.8b11384.
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This work was written as part of one of the author's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.
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Abstract
NMR has provided a wealth of structural and dynamical information for RNA molecules of up to ~50 nucleotides, but its application to larger RNAs has been hampered in part by difficulties establishing global structural features. A potential solution involves measurement of NMR perturbations after site-specific paramagnetic labeling. Although the approach works well for proteins, the inability to place the label at specific sites has prevented its application to larger RNAs transcribed in vitro. Here, we present a strategy in which RNA loop residues are modified to promote binding to a paramagnetically tagged reporter protein. Lanthanide-induced pseudocontact shifts are demonstrated for a 232-nucleotide RNA bound to tagged derivatives of the spliceosomal U1A RNA-binding domain. Further, the method is validated with a 36-nucleotide RNA for which measured NMR values agreed with predictions based on the previously known protein and RNA structures. The ability to readily insert U1A binding sites into ubiquitous hairpin and/or loop structures should make this approach broadly applicable for the atomic-level study of large RNAs.