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.

Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/Picarrier SCaMC1

“…As indicated by the diffraction pattern (Fig. 3), 2 M Li 2 SO 4 in the reservoir was sufficient to cryoprotect the crystals, similar to previous observations using 2 M NaCl as reservoir solution Yang, Faucher et al, 2011;Yang et al, 2010Yang et al, , 2013. Thus, no additional cryoprotectant solution was needed.…”

Crystal Structure of Human Poly(A) Polymerase Gamma Reveals a Conserved Catalytic Core for Canonical Poly(A) Polymerases

“…The coding region of hPAPγ was PCR amplified with specific primers and the resulting sequence was confirmed to correspond to the reference sequence NM_022894. The PCR product was cloned into a plasmid vector and residues 1-508 were subcloned into expression vector pGM10 with a His 6 tag at the N-terminus 54; 55 .…”

A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur

“…Purified Fur protein was dialyzed against buffer [50 mM Tris-Cl, 500 mM NaCl, 100 µM MnCl 2 , 10% glycerol, pH 7.9]. The 6xHis tag was cleaved using a biotinylated thrombin (Novagen) and removed by flowing through another Ni-charged resin [40] , [46] , [47] .…”