Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria

Supporting a co-translational model of protein import into mitochondria, we have previously shown that ribosome–nascent chain complexes (RNCs) specifically bind to mitochondria. When producing RNCs using the rabbit reticulocyte lysate in vitro translation system, it was necessary to maximize ribosome loading with truncated nascent proteins because it had a direct impact on RNC binding. We describe here the optimal conditions for preparing RNCs. We show that translation temperature and reaction time are two critical factors, with 30 °C and 15 min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.