On the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system

Abstract

As a test of the fidelity of the rabbit reticulocyte lysate system, we have examined the products of translation of various different influenza virus mRNAs, produced by in vitro transcription. A common finding with all mRNA species was that the ratio of full-length translation product to incomplete products decreased with increasing mRNA concentration. These short products are a mixture of (i) polypeptides initiated at the authentic initiation site but terminated prematurely, and (ii) polypeptides initiated at internal sites and terminated at the correct site. Analysis of mRNA stability during the translation assay showed very little degradation, quite insufficient to be the principle cause of incomplete product synthesis. Investigation of the influence of various parameters on the ratio of full-length to incomplete products leads to the conclusion that a high fidelity of translation can be obtained provided certain precautions are followed: the use of capped, rather than uncapped, mRNAs at low concentrations, with KCl concentrations about 20 mM above the level that gives maximum incorporation.