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 degrees Celsius and 15min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.
MacKenzie
James A
JA
Department of Biological Sciences, Oswego State University of New York, Oswego, NY 13126, USA.
Payne
R Mark
RM
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide