Use of Vaccinia Virus Poly(A) Polymerase for RNA 3'-End Labeling with a Chain-Terminating Nucleotide or a Short 3' Homopolymer Tract

摘要

Conditions are described for the 3'-end labeling of RNA with ~(32)P 3'-dATP (3'-de-oxyadenosine-5'-triphosphate), a chain-terminating nucleotide, using the poly(A) polymerase (PAP) encoded by vaccinia virus. Reaction time, divalent cation species andconcentration, and the requirement for both subunits of the PAP were investigated In the presence of Mn~(2+), vaccinia PAP is able to tail RNA primers with tracts of 3'-oligo(U), oligo(C) and oligo(G). Conditions for the addition of labeled 3'-homopolymer tracts were characterized. The use of low nucleotide concentrations in this study revealed an apparently fixed divalent cation concentration optimum of 0.1 mM, distinct from the previously noted requirement for a 1:1 divalent cation:NTP complex. This indicates a possible requirement for multiple divalent cations in nucleotidyl transfer by vaccinia PAP.