Poly(A) Polymerase from Vaccinia Virus-Infected Cells II. Product and Primer Characterization

The product of the in vitro reaction of a vaccinia virus-induced poly(A) polymerase (see preceding paper) with ATP is shown to be poly(A) by nuclease resistance and by annealing with poly(U). Polyacrylamide gel electrophoresis indicates that the in vitro synthesized poly(A) is associated with large RNA which is sensitive to RNase. RNA which co-purifies with the virus-induced enzyme is similar to vaccinia virus-specific RNA with respect to size and poly(A) content. Double labeling studies indicate that the RNA which co-purifies with the enzyme becomes associated with the poly(A) synthesized in vitro. The poly(A) formed in vitro is located on the 3′-OH terminus of this RNA. During in vitro poly(A) synthesis ³²P from α-[³²P]ATP is transferred to nucleosides other than 2′,3′-AMP, primarily to CMP. Inclusion of poly(U) in the in vitro reactions results in an increase in the transfer of ³²P to UMP.