Ambiguity in a Polypeptide-Synthesizing Extract from Saccharomyces cerevisiae

Abstract

Environmental factors known to induce ambiguity in bacterial extracts were tested in an in vitro cytoplasmic polypeptide-synthesizing system derived from Saccharomyces cerevisiae . Increasing concentrations of magnesium, spermine, and spermidine resulted in extensive leucine-phenylalanine ambiguity in polyuridylic acid-directed polypeptide synthesis. Kinetic studies showed that spermine-mediated stimulation of leucine incorporation occurred when phenylalanine was being actively incorporated. In addition to leucine, the amino acids isoleucine and serine were incorporated in the presence of added magnesium and spermine. Ambiguity in the presence of a high Mg 2+ concentration was decreased when the pH of the reaction mixture was lowered. Ethanol and neomycin enhanced ambiguity to a small, but significant, extent. Streptomycin and temperature had no effect on ambiguity. Leucine, isoleucine, and serine were not attached to phenylalanine transfer ribonucleic acid (tRNA) when the aminoacylation reaction was performed at increasing Mg 2+ and spermine concentrations. On the other hand, increasing levels of Mg 2+ and spermine stimulated the incorporation of leucine from tRNA into polypeptide during the transfer reaction. The formal similarity between the findings in the yeast and Escherichia coli systems implies the existence of a tRNA-screening site on the yeast ribosome comparable to that suggested for bacteria. A proposal is made as to the manner in which this site may function to produce the ambiguous codon translation observed.