Purification and properties of a ribonucleic acid primer-independent polyriboadenylate polymerase from Escherichia coli.

An enzyme isolated from Escherichia coli catalyzes the synthesis of polyriboadenylate from ATP in the absence of a RNA primer. The reaction is dependent on the presence of either Mg 2+ or Mn*+, 0.8 or 0.2 mu being optimal, respectively. Addition of Mg2f to reaction mixtures containing optimal concentration of Mn2+ or vice versa results in marked inhibition of the polymerization reaction. Inclusion of KC1 in the reaction stimulates the rate of polymerization, the optimum concentration being 0.16 M. The polymerization reaction is not inhibited by RNase or by polyriboadenylate and shows a lag period of 4 to 6 min and then proceeds linearly with time. The lag period can be removed by the addition of fresh enzyme to a reaction mixture inactivated by heat at 4 min. The lag in the polymerization reaction cannot be abolished by the addition of tRNA, DNAs, and oligoadenylates even though these polynucleotides enhanced the reaction rate by 50 to 70%. The increase in activity is not due to the formation of a covalent linkage between the product polyriboadenylate and either tRNA or DNA. The Km for ATP is 1.4 X lo+ M. Other ribonucleoside triphosphates (UTP, CTP, and GTP) do not serve as substrates. The guanine nucleotides, GTP, dGTP, and GDP, markedly decrease the rate of poly(A) synthesis from ATP while GMP failed to inhibit the reaction.