Synthesis of an Oligonucleotide Inhibitor of Protein Synthesis in Rabbit Reticulocyte Lysates Analogous to that Formed in Extracts from Interferon‐Treated Cells

Abstract

A heat-stable, low-MW inhibitor of protein synthesis is formed on incubation of hemin-supplemented rabbit reticulocyte lysates with ATP and double-stranded (ds)RNA. It inhibits the translation of added encephalomyocarditis virus RNA (EMCRNA) and endogeneous mRNA in reticulocyte lysates and mouse [fibroblast] L cell extracts. The enzyme responsible for the synthesis of the inhibitor binds to dsRNA and can be purified on a column of poly(I).cntdot.poly(C) bound to an inert support. The highly purified enzyme in its stable column-bound state can be conveniently employed to synthesize the inhibitor and to label it with [3H]ATP, or [.alpha.-32P]ATP or [.gamma.-32P]ATP as substrate. The radioactive inhibitor synthesized in this way with material from rabbit reticulocyte lysates shows the same spectrum of resistance and sensitivity to alkali and a variety of enzymes as corresponding material similarly synthesized with extracts from interferon-treated mouse L cells. The inhibitors from the 2 systems have comparable absorbance spectra, are chromatographically and electrophoretically indistinguishable and are apparently identical in specific activity in the inhibition of protein synthesis in the cell-free system. The inhibitor is also formed on inhibition of protein synthesis by dsRNA in reticulocyte lysates. On comparison of the spectrum of polypeptide products synthesized in response to EMC RNA in the reticulocyte lysate, the effects of the inhibitor or dsRNA were similar. A distinctly different effect was obtained with the hemin-controlled repressor, a known inhibitor of initiation. The significance of these results with respect to the mechanism of action of the inhibitor and its role in the inhibition observed in response to dsRNA is discussed.