Vaccinia virus poly(A) polymerase. Specificity for nucleotides and nucleotide analogs.
We have studied the nucleotide specificity of vaccinia virus poly(A) polymerase using a novel primer extension assay. Oligoribonucleotide primers labeled at the 5' end with 32P were elongated by the enzyme in the presence of ATP, leading to the 3' addition of greater than 1000 adenylate residues/primer molecule. In the presence of UTP, the enzyme catalyzed 3' polymerization of long poly(U) tails, albeit at a reduced rate of chain growth. In the presence of both ATP and UTP, 3' addition was selective for ATP. The transient accumulation of RNAs elongated by 10-16 residues suggested that polyadenylation (and polyuridylation) was a biphasic reaction. Quantitative 3' addition of GMP (from GTP) or CMP (from CTP) to the primer was also observed, although the rate of chain growth was so slow as to allow synthesis of only short oligo(G) or oligo(C) tails. The deoxynucleotides 3'-dATP (cordycepin triphosphate) and ddATP were markedly inhibitory to poly(A) polymerase. Primer elongation studies were consistent with inhibition due to 3' incorporation of inhibitor and chain termination. Incubation of enzyme with [alpha-32P] cordycepin triphosphate resulted in labeling of the Mr 57,000 enzyme subunit, apparently via formation of a covalent nucleotidyl-protein complex. These data are discussed in light of their implications for the catalytic mechanism of polyadenylation.
Shuman
S
S
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Moss
B
B
D000255:Adenosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014616:Vaccinia virus / Q000201:enzymology*
D009711:Nucleotides; D011061:Poly A; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase