Ribosomes: Methods for Preparation

Abstract

Ribosomes, the large protein–RNA complexes involved in translation of nucleotide sequences into proteins, are obtained from cells broken open by grinding, shear forces or enzymatically. They are purified from the cell lysate by centrifugation to remove cell debris, followed by sucrose density gradient centrifugation.

Keywords: ribosome; polysome; ribosomal subunits; sucrose density gradient

Figure 1.

Sucrose density gradient centrifugation. The sample containing ribosomes, ribosomal subunits or polysomes is layered on top of a buffer solution in which the concentration of sucrose increases from top to bottom in a centrifuge tube. The tubes are then spun in an ultracentrifuge in a horizontal position so that the centrifugal force is applied along the longitudinal axis of the tube. The heavier the particles, the farther they will move into the gradient. The centrifugation time depends on the types of particles to be separated. It is shortest for polysomes and longest for ribosomal subunits. After centrifugation, the gradient is fractionated and the A260 of the fractions is monitored.

Figure 2.

Typical A260 profiles obtained after sucrose density gradient centrifugation of (a) intact ribosomes, (b) ribosomal subunits, where the first peak corresponds to the small subunit, the second one to the large subunit, and (c) polysomes, where the first peak corresponds to monosomes (i.e. one ribosome attached to mRNA) and subsequent peaks to mRNA carrying two, three, etc. ribosomes. Note that the centrifugation time is different in each case (shortest for polysomes, longest for ribosomal subunits). The high absorbancy at the top of the gradient (left) is due to tRNA and free protein.

Figure 3.

Tandem affinity purification (TAP) of pre‐ribosomal particles. A cell extract is prepared from cells that have been genetically altered to express a specific, nonribosomal protein that is part of the pre‐ribosomal particle to be purified, linked to a ‘tag’. The latter consists of an epitope of Staphylococcus aureus protein A connected to the calmodulin‐binding peptide (CBP) via a cleavable linker peptide containing the consensus recognition site for the protease from tobacco etch virus (TEV). The extract is chromatographed on a column containing IgG‐Sepharose beads to bind the tagged particles. After washing, the particles are released by cleaving the linker peptide with the TEV protease. This exposes the CBP, allowing the tagged particles to bind to a calmodulin–Sepharose column in the presence of Ca2+ ions. Purification is completed by further washing and elution of the bound pre‐ribosomes using a buffer containing EGTA, which removes the Ca2+ ions required for binding to the column material.

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Further Reading

Bommer U, Burkhardt N and Jünemann R et al. (1997) Ribosomes and polysomes. In: Graham J and Rickwood D (eds) Subcellular Fractionation: A Practical Approach, pp. 271–301. Washington, DC: IRL Press.

Garrett RA, Douthwaite SR and Liljas A et al. (2000) The Ribosome: Structure, Function, Antibiotics and Cellular Interactions. Washington DC: American Society for Microbiology. [Conference proceedings.]

Matheson AT, Davies JE, Dennis PP and Hill WE (1995) Frontiers in translation. Biochemistry and Cell Biology 73: 739–1227.[Conference proceedings.].

Spector DL, Goldman RD and Leinwand LA (1998) Purification of ribosomes, ribosomal subunits, and polysomes. In: Cells: A Laboratory Manual, part II: Culture and Biochemical Analysis of Cells. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

The Ribosome, (2001) Cold Spring Harbor Symposia on Quantitative Biology. vol. LXVI Cold Spring Harbor: Cold Spring Harbor Press. [Conference proceedings.].

Tuite MF, Stansfield I and Planta RJ (1998) Identifying genes encoding components of the protein synthesis machinery of the yeast Saccharomyces cerevisiae. Methods in Microbiology 26: 351–373.

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How to Cite close
Raué, Hendrik A(May 2005) Ribosomes: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003964]