Poly(A) Polymerase, Yeast

Description:
Poly(A) Polymerase catalyzes the template independent of the addition of AMP from ATP to the 3´ end of RNA. Poly(A) works more competently than E. coli for RNA oligonucleotide-labeling and poly(A) tailing. Less incubation time is required for the yeast enzyme and labels both long and short substrates well. Poly(A) preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase. The reaction requires Mn²⁺ or Mg²⁺, ATP as substrate, and any RNA containing 3' hydroxyl termini as a primer. Longer RNA molecules are somewhat better primers than short oligomers. Substitution of cordycepin-5'-triphosphate (3'-dATP) for ATP results in the addition of a single 3'-dA residue to the ends of the RNA, a useful technique for labeling RNA at the 3' end.

Applications:

• Labeling the 3' ends of RNA with ATP or cordycepin
• Poly(A) tailing of RNA for cloning or affinity purification
• Preparing a priming site for cDNA synthesis using oligo-dT
• Enhancing translation of RNA transferred into eukaryotic cells

Source:
An E. coli strain that carries the cloned Poly(A) Polymerase gene from (Saccharomyces cerevisiae).

Specific Activity:
>20,000U/mg

Unit Definition:
One unit is the amount of enzyme which incorporates 1 pmol AMP into acid-insoluble material at 37°C in 1 min.

Storage Buffer:
20mM Tris-HCl (pH 8.0), 50mM KCl, 0.5mM DTT, 50% glycerol.

Heat Inactivation:
65°C for 20 minutes

References:

1. Sippel, A. E. (1973) Eur. J. Biochem. 37, 31-40.
2. Edmonds, M. (1982) in The Enzymes, 3^rd edition, ed. P. D. Boyer (Academic Press, New York) 15, 217-244.
3. Gething, M. J., Bye, J., Skehel, J. and Waterfield, M. (1980) Nature 287, 301-306.
4. Sano, H. and Feix, G. (1976) Eur. J. Biochem. 71, 577-583.