Regulation of protein synthesis in mammalian cells. V. Further studies on the effect of actinomycin D on translation control in Hela cells

The rate of protein synthesis in HeLa cells appears to be regulated, in part, by a factor which promotes the association of ribosomes with messenger RNA and whose production is inhibited by actinomycin. The decline in protein synthesis after the administration of actinomycin is not primarily due to a decay of available messenger RNA but, rather, is a result of a decrease in the rate of ribosomal association with message. The decay of protein synthesis in actinomycin can be varied over a wide range by altering the temperature of cell incubation. Thus the half-life of protein synthesis decay ranges from eight hours at 34 °C to two hours at 41°C. The rapid decline of protein synthesis at 41 °C is not accompanied by a corresponding decay of the messenger RNA. Polyribosomes decrease in size, but they can be restored to normal sedimentation distributions by low levels of cycloheximide, suggesting that messenger RNA remains functional. The translation rate at 41 °C is unaltered. The dose-response of protein synthesis inhibition by actinomycin was measured and a half-maximum inhibition was found to be effected by 0·1 μg of the drug/ml. Another important aspect of the regulation of translation in HeLa cells is the response of cells to depressed rates of protein synthesis. At 42 °C, protein synthesis is severely inhibited, due to a failure in the association of ribosomes with messenger RNA. Prolonged incubation at the elevated temperature results in a significant repair of the lesion. This repair is inhibited by actinomycin. The half-maximum inhibition is achieved at levels of from 0·05 to 0·1 μg of the drug/ml. The cell response to depressed rates of protein synthesis can also be demonstrated using the drug cycloheximide. Prolonged incubation in the drug results in a response which then can promote protein synthesis at 42 °C. Here again, the half-maximum inhibition of the response to cyclohemixide is achieved by 0·1 μg of actinomycin/ml. These experiments suggest, but do not prove, that the cellular response may be mediated through the synthesis of RNA that promotes the initiation of translation and does not involve the subsequent production of protein.