title: 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.
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.
authors.affiliation: Department of Microbiology and Molecular Genetics, Stafford Hall, University of Vermont, Burlington, Vermont 05405, USA.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yang
authors.forename: Frédérick
authors.initials: F
authors.lastname: Faucher
authors.forename: Molly
authors.initials: M
authors.lastname: Coseno
authors.forename: Joyce
authors.initials: J
authors.lastname: Heckman
authors.forename: Sylvie
authors.initials: S
authors.lastname: Doublié
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry*; D000818:Animals; D002021:Buffers; D002417:Cattle; D003432:Cross-Linking Reagents; D003460:Crystallization; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009843:Oligoribonucleotides / Q000737:chemistry*; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014961:X-Ray Diffraction
chemical_list: D000227:Adenine Nucleotides; D002021:Buffers; D003432:Cross-Linking Reagents; D004220:Disulfides; D009843:Oligoribonucleotides; D012333:RNA, Messenger; C023505:2',5'-oligoadenylate; D012313:RNA; D011062:Polynucleotide Adenylyltransferase