Stimulation of poly(A) tail elongation by the VP39 subunit of the vaccinia virus-encoded poly(A) polymerase.
The VP55 subunit of the vaccinia virus-encoded poly(A) polymerase can add a maximum of 35 adenylates to the 3'-end of an RNA primer in a rapid and highly processive manner, whereas the VP55-VP39 heterodimer catalyzes the formation of poly(A) tails several hundred nucleotides in length. Here, we describe the overexpression of the VP39 subunit, its purification to near homogeneity, and its ability to associate physically with VP55 and to stimulate polyadenylation. Although VP39 possessed no independent poly(A) polymerase activity, RNA primers with oligo(A) tails greater than 30 adenylates in length could be extended nearly 40-fold more rapidly in the presence of VP39. VP39 enhanced the polyadenylation rate by converting the slow, nonprocessive polyadenylation occurring after the rapid burst in the presence of monomeric VP55, to a rapid, semiprocessive reaction. The effect of VP39 was dramatic when poly(A) primers were used as, 60 mM NaCl, VP39 enhanced the polyadenylation rate 500-fold, and at 90 mM NaCl VP39 was absolutely required. Nevertheless, the VP39-containing polymerase remained selective for polyadenylation of an mRNA 3'-end in the presence of excess poly(A). These data suggest that the role of VP39 in polyadenylation is to increase the affinity of the polymerase for the growing poly(A) tail.
Gershon
P D
PD
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Moss
B
B
D017123:Bacteriophage T7 / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D010957:Plasmids; D011061:Poly A / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014162:Transfection; D014616:Vaccinia virus / Q000201:enzymology*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011061:Poly A; D012965:Sodium Chloride; D011062:Polynucleotide Adenylyltransferase