Uridylate-containing RNA sequences determine specificity for binding and polyadenylation by the catalytic subunit of vaccinia virus poly(A) polymerase.
VP55, the catalytic subunit of vaccinia virus poly(A) polymerase, has the remarkable property of adding 30-35 adenylates to RNA 3' ends in a rapid processive burst before an abrupt transition to slow, non-processive adenylate addition. Here, we demonstrate that this property results from the affinity of the enzyme for uridylate residues within the 3' 31-40 nt of the RNA primer. At physiological salt concentrations, both polyadenylation and stable VP55 binding required the presence of multiple uridylates within a 31-40 nt length of RNA, though specific RNA sequences were not necessary. Even DNA in which the deoxythymidylate residues were replaced with ribouridylates, could be polyadenylated in a processive manner. Both the unmethylated pyrimidine ring and a 2'-OH on the associated sugar are features of ribouridylates that are important for priming. The abrupt termination of processive polyadenylation was attributed to translocation of VP55 along the nascent poly(A) tail, which lacks uridylates for stable binding. As evidence for translocation and interaction with newly synthesized RNA, other homopolymer tails were synthesized by VP55 in the presence of Mn2+, which relaxes its donor nucleotide specificity. Only during poly(U) tail synthesis did processive nucleotide addition fail to terminate.
Gershon
P D
PD
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Moss
B
B
D001483:Base Sequence; D001692:Biological Transport; D002384:Catalysis; D006493:Heparin / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D012313:RNA; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012965:Sodium Chloride; D014542:Uridine Monophosphate / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology*
C034477:RNA primers; D012367:RNA, Viral; D011061:Poly A; D012965:Sodium Chloride; D012313:RNA; D006493:Heparin; D014542:Uridine Monophosphate; D011062:Polynucleotide Adenylyltransferase