Polysome profiling is a technique in

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

that is used to study the association of

mRNAs In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the p ...

with

ribosome Ribosomes () are molecular machine, macromolecular machines, found within all cell (biology), cells, that perform Translation (biology), biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order s ...

s. It is different from

ribosome profiling Ribosome profiling, or Ribo-Seq (also named ribosome footprinting), is an adaptation of a technique developed by Joan Steitz and Marilyn Kozak almost 50 years ago that Nicholas Ingolia and Jonathan Weissman adapted to work with next generation se ...

. 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

The procedure begins by making a

cell lysate Lysis ( ; from Greek language, Greek 'loosening') is the breaking down of the cell membrane, membrane of a Cell (biology), cell, often by virus, viral, enzyme, enzymic, or osmosis, osmotic (that is, "lytic" ) mechanisms that compromise its in ...

of the cells of interest. This lysate contains

polysomes A polyribosome (or polysome or ergosome) is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into po ...

, monosomes (composed of one ribosome residing on an

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...

), the small (40 S in

eukaryotes The eukaryotes ( ) constitute the domain of Eukaryota or Eukarya, organisms whose cells have a membrane-bound nucleus. All animals, plants, fungi, seaweeds, and many unicellular organisms are eukaryotes. They constitute a major group of ...

) and large (60S in eukaryotes) ribosomal subunits, "free" mRNA and a host of other

soluble In chemistry, solubility is the ability of a substance, the solute, to form a solution with another substance, the solvent. Insolubility is the opposite property, the inability of the solute to form such a solution. The extent of the solubi ...

cellular components. The procedure continues by making a continuous

sucrose Sucrose, a disaccharide, is a sugar composed of glucose and fructose subunits. It is produced naturally in plants and is the main constituent of white sugar. It has the molecular formula . For human consumption, sucrose is extracted and refined ...

gradient of continuously variable density in a

centrifuge A centrifuge is a device that uses centrifugal force to subject a specimen to a specified constant force - for example, to separate various components of a fluid. This is achieved by spinning the fluid at high speed within a container, thereby ...

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 Density (volumetric mass density or specific mass) is the ratio of a substance's mass to its volume. The symbol most often used for density is ''ρ'' (the lower case Greek letter rho), although the Latin letter ''D'' (or ''d'') can also be u ...

. A specific amount (as measured by

optical density Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative log ...

) 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 In physics, gravity (), also known as gravitation or a gravitational interaction, is a fundamental interaction, a mutual attraction between all massive particles. On Earth, gravity takes a slightly different meaning: the observed force b ...

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 ribosomes 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 ribosomes travel further still, and on and on. The "size" of the components is designated by S, the

svedberg In chemistry, a Svedberg unit or svedberg (symbol S, sometimes Sv) is a non- SI metric unit for sedimentation coefficients. The Svedberg unit offers a measure of a particle's size indirectly based on its sedimentation rate under acceleration ...

unit. Note that one S = 10⁻¹³ seconds, and that the concept of "big" is actually an oversimplification. Polysomesleft

After centrifugation, the contents of the tube are collected as fractions from the top (smaller, slower traveling) to bottom (bigger, faster traveling) and the optical density of the fractions is determined. The first fractions removed have a large amount of relatively small molecules, such as tRNAs, individual proteins, etc.

Applications

It is possible to use this technique to study the overall degree of translation in cells (for examples), but it can be used much more specifically to study individual proteins and their mRNAs. As an example shown in the lower portion of the figure, a protein that composes part of the small subunit can first be detected in the 40S fraction, then nearly disappears from the 60S fraction (the separations on these gradients are not absolute), then reappears in the 80S and polysome fractions. This indicates that there is at most very little of the protein found in the cell that is not part of the small subunit. In contrast, in the upper row of the immunoblot figure, a soluble protein appears in the soluble fractions and associated with ribosomes and polysomes. The particular protein is a

chaperone protein In molecular biology, molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are a number of classes of molecular chaperones, all of which function to assi ...

, which (in brief) helps to fold the nascent

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

as it is being extruded from the ribosome. As other work in the paper showed, there is a direct association of the chaperone with the ribosome. The technique can also be used to study the degree of translation of a particular mRNA In these experiments, 5' and 3' sequences of an mRNA were investigated for their effects on amount of mRNA produced and how well the mRNAs were translated. As shown, not all mRNA isoforms are translated with the same efficiency ''even though'' their coding sequences are the same.

References

{{reflist Molecular biology