Analysis of Translation Initiation During Stress Conditions by Polysome Profiling |
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Authors: | La?titia Coudert Pauline Adjibade Rachid Mazroui |
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Affiliation: | 1.Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Laval University;2.CHU de Quebec Research Center |
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Abstract: | Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions. |
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Keywords: | Cellular Biology Issue 87 Translation initiation polysome profile sucrose gradient protein and RNA isolation stress conditions |
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