Characterization of a polyriboadenylate polymerase from vaccinia virions.
A poly(A) polymerase with a molecular weight of approximately 80,000 containing 51,000 and 35,000 molecular weight subunits, was purified by affinity chromatography from vaccinia virus cores. The enzyme had a pH optimum of about 8.6, was dependent on divalent cations, and had considerably more activity with Mn-2+ than Mg-2+. At equimolar concentrations, other ribonucleoside triphosphates inhibited poly(A) polymerase activity by less than 10%; NaCl was extremely inhibitory at concentrations above 0.1 M. Under standard assay conditions, poly(A) polymerase activity was stimulated more than 10-fold by poly(C), but to small extent or not at all by other homopolyribonucleotides or natural RNA species unless they were first subjected to partial hydrolysis and alkaline phosphatase treatment. The ineffectiveness of most long polyribonucleotides was attributed to enzyme binding to internal regions. Short poly- or oligoribonucleotides prepared from natural or synthetic RNAs, except poly(G), exhibited similar priming abilities, and isotope transfer experiments indicated the covalent attachment of poly(A) to cytidylate, uridylate, and inosinate residues. Experiments with a series of uridylate oligomers indicated that the minimum effective primer length was four to six nucleotides. Partially digested DNA and short poly- and oligodeoxyribonucleotides of dT, dC, and dI, but not of dA and dG, also acted as effective primers for the poly(A) polymerase.
D000227:Adenine Nucleotides; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D003840:Deoxycholic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009994:Osmolar Concentration; D011119:Polynucleotides / Q000096:biosynthesis; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014616:Vaccinia virus / Q000201:enzymology*
D000227:Adenine Nucleotides; D011119:Polynucleotides; D003840:Deoxycholic Acid; D008345:Manganese; D012965:Sodium Chloride; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium