Polyadenylate polymerase from vaccinia virions.

Abstract

HETEROGENEOUS nuclear, messenger1–9 and viral1,10–16 RNAs from eukaryotic organisms contain long polyadenylate (poly (A)) sequences of unknown function. The poly (A) is located at the 3′ terminus17–21 and may be added after transcription is completed11,22. Although enzymes that can add adenylate residues to RNA have been obtained from eukaryotic organisms28–29, they have not yet been coupled with RNA synthesis in vitro and it is not known whether specific sequences in DNA or nascent RNA act as signals for the addition of adenylate residues. Vaccinia virions seem to be a uniquely suitable system for studying the biosynthesis de novo of poly (A) and its attachment to RNA. Kates and Beeson1,10 showed that isolated vaccinia virus cores synthesized poly (A) in the presence of ATP and that RNA with terminal poly (A) sequences was made in the presence of all four ribonucleoside triphosphates. They suggested1,10 that in this system the adenylate residues were added by a transcriptional mechanism, possibly by the viral DNA-dependent RNA polymerase30,31. Evidence for such a mechanism was indirect and included the finding that poly (A) synthesis was inhibited by ethidium bromide and proflavine, drugs that bind to double stranded nucleic acids by intercalation1,10. The possibility that vaccinia DNA contains poly (dA>poly (dT) tracts that could serve as templates was consistent with hybridization data and with the transcription of poly (A) sequences by E. coli RNA polymerase1,10. The potential usefulness of vaccinia virus enzymes, for the study of RNA and poly (A) synthesis, has not been fully realized because of the particulate nature of the viral core. Of the five enzymatic activities known to be present in vaccinia cores30–35, only the DNA-dependent nucleotide phosphohydrolase (ATPase) has been solubilized36. We now describe the solubilization, isolation and some interesting properties of the poly (A) polymerase from vaccinia virions.