On the translational control of histone synthesis. Quantitation of biologically active histone mRNA from synchronized HeLa cells and its translation in different cell-free systems.

Abstract

Histone mRNA was isolated from polyribosomes and other cell fractions of synchronized HeLa cells and quantitated by its translational capacity in a rabbit reticulocyte lysate. Inhibition of DNA synthesis with hydroxyurea (5 mM) results within 30 to 60 min in a loss of 80–85% of biologically active histone mRNA from polyribosomes. In contrast, actinomycin D (5 μg/ml) during the first hour of its action, only stops further accumulation of histone mRNA on polyribosomes in early S‐phase cells. Under conditions of interruption of DNA replication biologically active histone mRNA does not appear in other cell fractions suggesting that it is immediately destroyed in the cytoplasm.The possible translational control of histone synthesis in HeLa cells was investigated with special consideration of the existence of messenger‐specific initiation factors. Cell‐free extracts from synchronized HeLa cells in which DNA and histone synthesis were inhibited with hydroxyurea, fully retain their capacity to translate added homologous histone mRNA as compared with extracts from synchronized S‐phase cells. Using a partially purified protein‐synthesizing system from rat liver supplemented with initiation factors from synchronized HeLa cells differing in their histone‐synthesizing capacity, it was shown that factors from hydroxyurea‐treated cells are as active in stimulating the translation of histone mRNA, globin mRNA and tobacco mosaic virus RNA as are initiation factors from S‐phase cells. The results suggest that messenger discriminating translation factors are not involved in the regulation of histone synthesis in HeLa cells or they are very labile and inactivated during preparation.