Faithful and efficient translation of homologous and heterologous mRNAs in an mRNA-dependent cell-free system from Saccharomyces cerevisiae

Abstract

A cell-free protein synthesizing system from the yeast Saccharomyces cerevisiae has been optimized for the translation of both homologous yeast mRNA and for a number of heterologous eukaryotic mRNAs. A significant increase in protein synthesis was observed when K(OAc) rather than KCl was used as the source of K+ in the in vitro translation system. This was due primarily to an inhibitory effect oif Cl-. The polyamine putrescine hydrochloride stimulated protein synthesis only at low Mg2+ concentrations. Protein synthesis directed by both yeast mRNA and several eukaryotic mRNAs examined in the system was sensitive to the mRNA 5'-cap analogue, 7-methylguanosine 5'-monophosphate. One-dimensional and two-dimensional polyacrylamide gel analysis of polypeptides synthesized in response to yeast polysomal RNA demonstrated faithful translation in vitro. Translational control and post-translational modifications appear to operate normally in vitro. RNA from several eukaryotic viruses (brome mosaic virus, turnip yellow mosaic virus, and tobacco mosaic virus) were found to be faithfully translated in vitro yielding discrete polypeptides. Reticulocyte polysomal RNA directed the synthesis of a single protein that co-migrated with rabbit globin. The prokaryotic RNAs of Q beta and MS2 were translated with a very low efficiency. The yeast cell-free system programmed with yeast polysomal RNA provides an excellent model for the study of translational control in a eukaryote.