Purification crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine poly(A) polymerase and a chemically modified 15-mer oligo(A) RNA

摘要

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.