Abstract
VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites. Here, we show that the cognate RNA sequence comprises a 5'-rU sub(2)-N sub(15)-rU-3' motif (where N = any deoxyribo or ribonucleotide), embedded within oligonucleotide primers 29-30 nucleotides (nt), or greater, in length. Nine residues separate the 3'-most ribouridylate of the optimally positioned motif from the primer 3'-OH. A ribose sugar at the extreme 3'-terminal nucleotide of the primer is absolutely required for VP55's adenylyltransferase activity, but not for stable VP55-RNA interaction. A ribose at position -3 markedly stimulates both adenylyltransferase activity and stable binding. The use of uridine analogs indicated (i) those functional groups of the uracil base which contribute to stable VP55-primer interaction, and (ii) that VP55's ability to discriminate uracil from cytosine stems largely from the requirement for a protonated N3 nitrogen within the pyrimidine ring. The rU sub(2)-N sub(15)-rU motif was identified within the uridylate-rich 3' end of a naturally occurring vaccinia mRNA. However, oligonucleotides whose only internal uridylates comprised the motif supported only a 3-5-nt processive burst of oligo(A) tail addition, as opposed to the similar to 30-35-nt burst observed with the naturally occurring 3' end.