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Updated: Jun 4, 2026
Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker
Published on: January 3, 2019
Purification, crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine
Qin Yang1, Frédérick Faucher, Molly Coseno
1Department of Microbiology and Molecular Genetics, Stafford Hall, University of Vermont, Burlington, Vermont 05405, USA.
Abstract:
Poly(A) polymerase (PAP) synthesizes the polyadenine tail at the 3'-end of messenger RNA. A disulfide cross-linking strategy was implemented to obtain a complex between bovine PAP (bPAP) and a 15-mer oligo(A). All seven endogenous cysteines were mutated to eliminate nonspecific cross-linked complexes. A cysteine residue was introduced at several different positions and A152C was found to achieve maximum specific cross-linking efficiency. The resulting bPAP construct was active and, when mixed with a chemically modified RNA, yielded crystals of a bPAP-RNA complex. The crystals, which belonged to space group P2 and harbored two protein-RNA complexes per asymmetric unit, diffracted X-rays to 2.25 Å resolution.
