Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria
James A. MacKenzie, R. Payne
Cardiac Developmental Biology
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.
https://doi.org/10.1016/j.mito.2006.01.001
In vitro translation
Protein targeting
Ribosome-nascent chain complex
MacKenzie, J. A., & Payne, R. (2006). Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria. Mitochondrion, 6(2), 67-75. https://doi.org/10.1016/j.mito.2006.01.001
Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria. / MacKenzie, James A.; Payne, R.
In: Mitochondrion, Vol. 6, No. 2, 04.2006, p. 67-75.
MacKenzie, JA & Payne, R 2006, 'Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria', Mitochondrion, vol. 6, no. 2, pp. 67-75. https://doi.org/10.1016/j.mito.2006.01.001
MacKenzie JA, Payne R. Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria. Mitochondrion. 2006 Apr;6(2):67-75. https://doi.org/10.1016/j.mito.2006.01.001
MacKenzie, James A. ; Payne, R. / Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria. In: Mitochondrion. 2006 ; Vol. 6, No. 2. pp. 67-75.
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title = "Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria",
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 °C and 15 min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.",
keywords = "In vitro translation, Mitochondria, Protein targeting, Ribosome-nascent chain complex, Ribosomes",
author = "MacKenzie, {James A.} and R. Payne",
doi = "10.1016/j.mito.2006.01.001",
journal = "Mitochondrion",
T1 - Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria
AU - MacKenzie, James A.
AU - Payne, R.
N2 - 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.
AB - 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.
KW - In vitro translation
KW - Protein targeting
KW - Ribosome-nascent chain complex
KW - Ribosomes
U2 - 10.1016/j.mito.2006.01.001
DO - 10.1016/j.mito.2006.01.001
JO - Mitochondrion
JF - Mitochondrion
10.1016/j.mito.2006.01.001