Accuracy of in vitro protein synthesis: translation of polyuridylic acid by cell-free extracts of human fibroblasts.

Overview

Errors in translation have been measured in cell-free protein synthesising extracts derived from cultured MRC-5 human diploid fibroblasts of limited lifespan. Using polyuridylic acid as messenger, the error frequency for the misincorporation of leucine was 1-2%. It was found that varying the concentration of leucine increased the error frequency for leucine misincorporation. No difference could be detected in the accuracy of translation with increasing cell age, from passage 25 to 55. The aminoglycoside antibiotic, paromomycin, was shown to have a profound effect on the leucine misincorporation, increasing the error frequency of this amino acid twenty-to fortyfold. However, there was no difference in the paromomycin-induced errors wtih increasing cell age. Another effect of this antibiotic is that it inhibits the incorporation of the cognate amino acid phenylalanine. It was found that passage 55 cell extracts were less inhibited by paromomycin than similar extracts made from lower passage cells (passages 25 and 40). When the accuracy of translation of cell extracts made from human transformed cells (HeLa) and untransformed cells (MRC-5) was compared, no detectable difference could be found. Paromomycin increased the leucine errors in extracts made from HeLa cells to a similar degree to that observed for MRC-5 fibroblasts.