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

Mitochondrion
James A MacKenzie, R M Payne

Abstract

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 degrees Celsius and 15min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.

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Citations

May 28, 2010·Plant Molecular Biology·Morgane MichaudAnne-Marie Duchêne
Apr 8, 2015·Drug Development Research·Matthew V CannonCarl A Pinkert
Mar 25, 2006·Journal of Experimental Botany·David C Logan
Feb 28, 2021·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Tianhong SuMing Kuang

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