Studies on protein synthesis in eukaryotic subcellular systems

Abstract

Summaryo1Polyphenylalanine is synthesized in a cell-free system from yeast after attachment of free ribosomes to polyuridylic acid. A maximum activity was found at 16–20 mM MgCl2. On the other hand, at 8 mM MgCl2, phenylalanine is incorporated into preexisting nascent peptide chains under the direction of polyuridylic acid. This synthesis is brought about by release of ribosomes from their original messenger and their subsequent attachment to poly-U. For this exchange reaction the acceptor- (or A-) site of the ribosome should be vacant.2.Yeast polysomes were found to dissociate in 0.5 M KCl into ribosomal subunits, smaller polysomes and monosomes to an extent depending on the duration of incubation rich medium of the protoplasts from which they were derived. Ribosomes within polysomes that dissociate into subunits in 0.5 M KCl seem to have participated normally in protein synthesis, the larger subunit carrying the peptidyl-tRNA after dissociation. Ribosomes within polysomes that are resistant to dissociation in 0.5 M KCl remain so after the completion in vitro of one or more elongation cycles, but dissociate in 0.5 M KCl after puromycin treatment. The reason why some ribosomes within polysomes dissociate in 0.5 M KCl has not yet been clarified.3.A preliminary account is given of a cell-free system in which polysomes from brain tissue of neonatal mice are highly active (up to 200 amino acids per ribosome) in endogenous protein synthesis. The system is apparently deficient in release and/or termination as well as in re-initiation. A labelled product of the system has been isolated which co-migrates with the microtubule protein during disk gel electrophoresis. Polyphenylalanine is synthesized in a cell-free system from yeast after attachment of free ribosomes to polyuridylic acid. A maximum activity was found at 16–20 mM MgCl2. On the other hand, at 8 mM MgCl2, phenylalanine is incorporated into preexisting nascent peptide chains under the direction of polyuridylic acid. This synthesis is brought about by release of ribosomes from their original messenger and their subsequent attachment to poly-U. For this exchange reaction the acceptor- (or A-) site of the ribosome should be vacant. Yeast polysomes were found to dissociate in 0.5 M KCl into ribosomal subunits, smaller polysomes and monosomes to an extent depending on the duration of incubation rich medium of the protoplasts from which they were derived. Ribosomes within polysomes that dissociate into subunits in 0.5 M KCl seem to have participated normally in protein synthesis, the larger subunit carrying the peptidyl-tRNA after dissociation. Ribosomes within polysomes that are resistant to dissociation in 0.5 M KCl remain so after the completion in vitro of one or more elongation cycles, but dissociate in 0.5 M KCl after puromycin treatment. The reason why some ribosomes within polysomes dissociate in 0.5 M KCl has not yet been clarified. A preliminary account is given of a cell-free system in which polysomes from brain tissue of neonatal mice are highly active (up to 200 amino acids per ribosome) in endogenous protein synthesis. The system is apparently deficient in release and/or termination as well as in re-initiation. A labelled product of the system has been isolated which co-migrates with the microtubule protein during disk gel electrophoresis.