Purification and characterization of full-length mammalian poly(A) polymerase.
Bovine poly(A) polymerase was purified from overexpressing strains of Escherichia coli and from Spodoptera frugiperda Sf21 cells infected with a recombinant baculovirus. The E. coli-expressed enzyme had an apparent molecular mass of 85 kDa in SDS gels, as anticipated from the cDNA sequence. Poly(A) polymerase from insect cells consisted of several species with higher apparent molecular weights due to phosphorylation. The two preparations showed minor differences in their catalytic properties. The insect cell-expressed enzyme had a 5-fold higher Km for the primer in a nonspecific Mn(2+)-dependent polyadenylation reaction and a lower activity in specific AAUAAA-dependent polyadenylation and generated shorter poly(A) tails during the processive phase of polyadenylation. Both recombinant poly(A) polymerases stimulated 3'-cleavage of the SV40 late mRNA precursor. Neither preparation contained ATPase or poly(A) degrading activity. The enzyme polymerized adenosine 5'-O-(1-thiotriphosphate), SP-diastereomer, with inversion of configuration. Thus, poly(A) synthesis proceeds via an SN2-in-line mechanism without covalent intermediate.
Wittmann
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T
Department of Cell Biology, Biozentrum, University of Basel, Switzerland.
Wahle
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003435:Crotalid Venoms / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D010766:Phosphorylation; D039102:Poly(A)-Binding Proteins; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013539:Simian virus 40; D018411:Spodoptera; D013237:Stereoisomerism; D013873:Thionucleotides / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors
D003435:Crotalid Venoms; D039102:Poly(A)-Binding Proteins; D012322:RNA Precursors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D039221:mRNA Cleavage and Polyadenylation Factors; D008345:Manganese; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase; D010727:Phosphoric Diester Hydrolases; D008274:Magnesium