Yeast protein synthesis. Preparation and analysis of a highly active cell-free system

Abstract

A detailed description is given of the techniques for preparing, handling and assaying a cell-free protein-synthesizing system from yeast, analogous to crude (S-30) Escherichia coli extracts. Its basic characteristics are described. The rate of poly(U)-directed polyphenylalanine synthesis was at least fivefold higher than in previously reported yeast cell-free systems, approaching that of crude mammalian cell-free systems. Fractionation of the S-30 extracts lowered activity. Organelles and their fragments present in the S-30 extract neither contributed to nor inhibited cytoplasmic protein synthesis. There was a component localized in the high-speed supernatant that caused an inhibition of polyphenylalanine synthesis. Poly(U) programmed the synthesis of long-chain polyphenylalanine, in contrast with the only other yeast system in which this has been examined (Bretthauer & Golichowski, 1968). Preincubation techniques inactivated the system and probably a small proportion only of the ribosomes was active.