Specific recognition of an rU2-N15-rU motif by VP55, the vaccinia virus poly(A) polymerase catalytic subunit.
VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites (Deng, L., and Gershon, P. D. (1997) EMBO J. 16, 1103-1113). Here, we show that the cognate RNA sequence comprises a 5'-rU2-N15-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 rU2-N15-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 approximately 30-35-nt burst observed with the naturally occurring 3' end.
Deng
L
L
Institute of Biosciences and Technology/Department of Biochemistry and Biophysics, Texas A&M University, Houston, Texas 77030-3303, USA.
Beigelman
L
L
Matulic-Adamic
J
J
Karpeisky
A
A
Gershon
P D
PD
D001665:Binding Sites; D008956:Models, Chemical; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D014498:Uracil / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology*
C034477:RNA primers; D012333:RNA, Messenger; D012367:RNA, Viral; D014498:Uracil; D012313:RNA; D012266:Ribose; D014542:Uridine Monophosphate; D011062:Polynucleotide Adenylyltransferase