More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.
- MeSH
- centrifugace - gradient hustoty metody MeSH
- elektroforéza metody MeSH
- interpretace statistických dat MeSH
- kvasinky MeSH
- polyribozomy chemie MeSH
- proteosyntéza genetika MeSH
- regulace genové exprese genetika MeSH
- RNA izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
