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 wi th a recombinant baculovirus. The E. coli-expressed enzyme had an appa rent molecular mass of 85 kDa in SDS gels, as anticipated from the cDN A 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 prop erties. The insect cell-expressed enzyme had a 5-fold higher K-m for t he primer in a nonspecific Mn2+-dependent polyadenylation reaction and a lower activity in specific AAUAAA-dependent polyadenylation and gen erated shorter poly(A) tails during the processive phase of polyadenyl ation. 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-t hiotriphosphate), S-P-diastereomer, with inversion of configuration. T hus, poly(A) synthesis proceeds via an S(N)2-in-line mechanism without covalent intermediate.