Transition from rapid processive to slow nonprocessive polyadenylation by vaccinia virus poly(A) polymerase catalytic subunit is regulated by the net length of the poly(A) tail

Abstract

The mRNA of vaccinia virus, like that of eukaryotes, possesses a poly(A) tail. VP55, the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was overexpressed and purified to near homogeneity. VP55 polyadenylated a 30-mer primer representing the 3′ end of a vaccinia virus mRNA bimodally: 30-35 adenylates were added in a rapid, processive, initial burst, after which polyadenylation decelerated dramatically and became nonprocessive. Polyadenylation of variants of the 30-mer primer, which contained preformed 3′-oligo(A) extensions, showed that the transition between the two modes of polyadenylation was regulated by the net length of the 3′-oligo(A) tail rather than the number of adenylate additions catalyzed by VP55. Primers comprising oligo(A) alone were polyadenylated only if they were >34 nucleotides in length and, then, only in the slow nonprocessive mode. These data support a dynamic model whereby the mode of polyadenylation by VP55 is regulated by sequences within the 3′ 30-35 nucleotides of the mRNA: Polyadenylation is rapid and processive until a net 3′-oligo(A) length of 30-35 nucleotides is achieved. Consistent with this, excess oligo(A) did not compete with the 30-mer primer for rapid processive polyadenylation. The primer specificity of VP55 may contribute to the selective polyadenylation of newly formed mRNA.