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Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.
Dieter A Wolf1, Jürg Bähler2, Jo Ann Wise3
1Degenerative Disease Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037; dwolf@sbpdiscovery.org j.bahler@ucl.ac.uk.
Cold Spring Harbor Protocols
|April 5, 2017
Summary
This study details a polysome profiling method using ultracentrifugation to separate ribosomal subunits and complexes. This technique enables detailed analysis of translation regulation and RNA populations.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Translation is a fundamental biological process crucial for gene expression.
- Understanding translation regulation is key to deciphering cellular mechanisms.
- Polysome profiling is a standard technique for studying translation.
Purpose of the Study:
- To describe a robust method for polysome profile analysis.
- To enable the separation and characterization of ribosomal complexes.
- To facilitate the study of translation regulation.
Main Methods:
- Whole cell extracts are prepared and subjected to high-velocity ultracentrifugation on linear sucrose gradients.
- Cycloheximide is used to stabilize polysomes by inhibiting translation.
- Fractions are collected and analyzed for RNA and protein content.
Main Results:
- Separation of 40S and 60S ribosomal subunits, 80S monosomes, and polysomes.
- Enables downstream analysis of specific RNA and protein species.
- Facilitates comprehensive profiling of the transcriptome and proteome involved in translation.
Conclusions:
- This polysome profiling method provides a powerful tool for investigating translation.
- It allows for detailed analysis of translational control mechanisms.
- The technique supports diverse downstream applications including RNA sequencing and mass spectrometry.