Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid

Total RNA was isolated from rat liver polyribosomes and fractionated by oligo(dT)-cellulose chromatography to obtain polyadenylated mRNA. The mRNA was translated in a wheat-germ cell-free protein-synthesizing system containing 3Hglycine, 3Hlysine and 3Hserine. Most of the newly synthesized 3H-labelled polypeptides were removed from the cell-free products by precipitation at pH 4.0. 3H-labelled thionein chains, which were soluble at pH 4.0, were purified by activated-thiol-Sepharose 4B chromatography or by gel-filtration chromatography. Polyribosomal thionein mRNA was found to increase by at least 3-fold after parenteral administration and by 20 h thereafter the ratio of thionein mRNA to total mRNA approached that found in controls. Actinomycin D administration in vivo blocked the Zn2+-induced increase in polyribosomal thionein mRNA content. These data strongly suggest that metallothionein is an inducible protein. The mechanism of regulation appears to involve changes in the synthesis de novo of thionein mRNA and hence the pool of thionein mRNA available for translation.