title: Specific recognition of an rU2-N15-rU motif by VP55, the vaccinia virus poly(A) polymerase catalytic subunit.
abstract: 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.
authors.affiliation: Institute of Biosciences and Technology/Department of Biochemistry and Biophysics, Texas A&M University, Houston, Texas 77030-3303, USA.
authors.forename: L
authors.initials: L
authors.lastname: Deng
authors.forename: L
authors.initials: L
authors.lastname: Beigelman
authors.forename: J
authors.initials: J
authors.lastname: Matulic-Adamic
authors.forename: A
authors.initials: A
authors.lastname: Karpeisky
authors.forename: P D
authors.initials: PD
authors.lastname: Gershon
mesh_terms: 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*
chemical_list: C034477:RNA primers; D012333:RNA, Messenger; D012367:RNA, Viral; D014498:Uracil; D012313:RNA; D012266:Ribose; D014542:Uridine Monophosphate; D011062:Polynucleotide Adenylyltransferase