This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.
1. Preparation of 7-47% sucrose gradients
Final Concentration | 7% sucrose | 47% sucrose |
7% | 48 mL | 0 |
17% | 36 mL | 12 mL |
27% | 24 mL | 24 mL |
37% | 12 mL | 36 mL |
47% | 0 | 48 mL |
2. Preparation of the yeast extract
3. Preparation of extracts from mammalian cells
4. Centrifugation of gradients
5. Collection of data and fractions
6. Representative Results
Figure 1. Representative trace of ribosome extract prepared from yeast strain BY4741 (mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) in the presence of cycloheximide. The ribosomal extract was fractionated utilizing a 7 47% sucrose gradient and analyzed using a pump syringe apparatus attached to a UV detector. Peaks containing polyribosomes and 80S, 60S and 40S ribosomes are indicated.
The information obtained from the polyribosome profile can provide valuable insight into the translational status of the cell. In addition, the status of the assembly of ribosomal subunits themselves can be determined2. For example the presence of halfmers or 80S and larger peaks with a slight shoulder to the right on the profile indicates a bound 40S subunit awaiting 60S subunit joining. Performing the experiment in the absence of any added cycloheximide or other inhibitor of elongation allows for analysis of the rate of run off, which indicates whether or not elongation is altered3. The fractions themselves are a rich source of reagents for the subsequent determination of the association of a specific mRNA or protein with ribosomal subpopulations by Northern or Western blotting of the fractions. The total pool of mRNAs associated with active ribosomes can be determined via an associated microarray4 or deep sequencing analysis5. The protein associations can also be stabilized by the appropriate addition of a cross linking reagent 6.
Polyribosome analysis from mammalian cells is also worth mentioning as a distinct protocol in terms of the apparent difficulties in establishing the conditions required to obtain stable polysomes. The buffer systems reported in the literature are widely variable 7-10, thus there may be a requirement for cell-type specific optimization of the lysis buffer, or it is equally plausible that the buffer system is not as important as keen attention to working with fresh lysates kept at low temperatures. To our knowledge a systematic evaluation of the parameters affecting mammalian polysome formation has not been reported.
The authors have nothing to disclose.
The authors would like to acknowledge John Woolford for the initial basis of this method, TGK is supported by NIH GM057483 and AI076245 and PRC is supported by GM77073.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
polyallomer ultracentrifuge tube | Beckman | 326823 | 1 x 3.5 in. (25x89mm) | |
Fluorinert | Sigma | F9755 | ||
Cycloheximde | Sigma | C-7698 | ||
Heparin | Sigma | H3393 | ||
BY4741 | Open Biosystems | YSC1048 | Other strains work equally well |