Synthesis of an oligonucleotide inhibitor of protein synthesis in rabbit reticulocyte lysates analogous to that formed in extracts from interferon-treated cells.
A heat-stable, low-molecular-weight inhibitor of protein synthesis is formed on incubation of haemin-supplemented rabbit reticulocyte lysates with ATP and double-stranded RNA (dsRNA). It inhibits the translation of both added encephalomyocarditis virus RNA (EMC RNA) and endogeneous messenger RNA in reticulocyte lysates and mouse 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).poly (C) bound to an inert support. The highly purified enzyme in its stable column-bound state can be conveniently employed to synthesise the inhibitor and to label it with [3H]ATP, or [alpha-32P]ATP or [gamma-32P]ATP as substrate. The radioactive inhibitor synthesised 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 synthesised with extracts from interferon-treated mouse L-cells. The inhibitors from the two 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 synthesised 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 haemin-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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System; D004686:Encephalomyocarditis virus / Q000378:metabolism; D004798:Enzymes / Q000302:isolation & purification; D006427:Hemin / Q000378:metabolism; D007372:Interferons; D007739:L Cells / Q000378:metabolism; D009841:Oligonucleotides / Q000096:biosynthesis*; D009843:Oligoribonucleotides / Q000096:biosynthesis*; D014176:Protein Biosynthesis / Q000187:drug effects; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D004798:Enzymes; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012313:RNA; D006427:Hemin; D000255:Adenosine Triphosphate; D007372:Interferons