Association of poly(A) and poly(U) polymerases with cytoplasmic ribosomes.

Abstract

Cytoplasmic ribosomes of Ehrlich ascites tumour cells were isolated under conditions where ribonuclease contamination is minimized. These ribosomes displayed poly(A) and poly(U) polymerase activity with [3H]ATP or [3H]UTP as substrates, while incorporation of [3H]GTP or [3H]CTP was not observed. Poly(A) polymerase is Mn2+ ‐dependent with an optimum at 1 mM, while poly(U) polymerase is activated by Mg2+ with a broad optimum at 5–20 mM. Both enzyme activities remained associated with the two ribosomal subparticles upon EDTA treatment and sucrose density gradient fractionation. The product of poly(U) polymerase was bound to endogenous 28‐S and 18‐S rRNA. About 40% of the poly(A) polymerase product was retained by oligo(dT)‐cellulose and the radioactivity distributed in 28‐S and 18‐S rRNA as well as in 4–16‐S RNA material. Addition of poly(A) stimulates markedly the activity of poly(A) polymerase, but addition of poly(U) is without effect on either enzyme. The poly(U) polymerase of the small and the large subparticle did not display a preferential use of endogenous versus exogenous RNA as primer. The same was true for poly(A) polymerase of the small subparticle. However, the poly(A) polymerase of the large subparticle showed a marked preference for exogenous cytoplasmic 18‐S RNA before or after purification through oligo(dT)‐cellulose. This effect was not observed with 18‐S rRNA obtained from previously isolated small ribosomal subparticles. It is suggested that ribosome‐associated poly(A) polymerase may display a preference for mRNA lacking poly(A) as primer.