Polysome Profiling

Polysome profiling is a technique in molecular biology that is used to study the association of mRNAs with ribosomes. It is important to note that this technique is different than ribosome profiling. Both techniques have been reviewed and both are used in analysis of the translatome, but the data they generate are at very different levels of specificity. When employed by experts, the technique is remarkably reproducible: the 3 profiles in the first image are from 3 different experiments.

The procedure begins by making a cell lysate of the cells of interest. This lysate contains polysomes, monosomes (composed of one ribosome residing on an mRNA), the small (40S in eukaryotes) and large (60S in eukaryotes) ribosomal subunits, "free" mRNA and a host of other soluble cellular components.

The procedure continues by making a continuous sucrose gradient of continuously-variable density in a centrifuge tube. At the concentrations used (15-45% in the example), sucrose does not disrupt the association of ribosomes and mRNA. The 15% portion of the gradient is at the top of the tube, while the 45% portion is at the bottom because of their different density.

A specific amount (as measured by optical density) of the lysate is then layered gently on top of the gradient in the tube. The lysate, even though it contains a large amount of soluble material, is much less dense than 15% sucrose, and so it can be kept as a separate layer at the top of the tube if this is done gently.

In order to separate the components of the lysate, the preparation is subjected to centrifugation. This accelerates the components of the lysate with many times the force of gravity and thus propels them through the gradient based upon how "big" the individual components are. The small (40S) subunits travel less far into the gradient than the large (60S) subunits. The 80S ribsomes on an mRNA travel further (note that the contribution of the size of the mRNA to the distance traveled is not significant). Polysomes composed of 2 ribosomes travel further, polysomes with 3 ribsomes travel further still, and on and on. The "size" of the components is designated by S, the svedberg unit. Note that one S = 10⁻¹³ seconds, and that the concept of "big" is actually an oversimplification.

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