Cell‐Free Translation and Regulation of Candida lipolytica Acetyl‐Coenzyme‐A Carboxylase Messenger RNA

Abstract

Messenger(m)RNA from C. lipolytica directs the synthesis of complete acetyl-CoA carboxylase in the mRNA-dependent reticulocyte lysate cell-free translation system. The identity of the translation product is evidenced by the following results: first, it is immunoprecipitated with antibody to acetyl-CoA carboxylase and competes with authentic acetyl-CoA carboxylase for binding to the antibody; second, it comigrates with authentic acetyl-CoA carboxylase upon dodecylsulfate/polyacrylamide gel electrophoresis; finally, the peptide fragments formed by its partial proteolysis with papain or .alpha.-chymotrypsin are identical with those formed from authentic acetyl-CoA carboxylase. Using this assay system, it was demonstrated that the level of acetyl-CoA carboxylase mRNA activity in C. lipolytica cells decreases with increasing concentrations of oleic acid in culture medium and that the changes in the mRNA activity parallel those in the cellular level of acetyl-CoA carboxylase. This finding, in conjunction with a previous study, indicates that the diminished synthesis of acetyl-CoA carboxylase in cells grown in the presence of fatty acid is due to a reduced level of the mRNA coding for the enzyme.