Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.
Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.
1. Preparation of a cysteine charged Sulfolink resin
2. Chromatographic purification of ribosomes using a cysteine charged Sulfolink resin
Note: If working with a strain where protease activity proves to be an issue, protease inhibition cocktails can be used in all subsequent buffers.
3. Puromycin treatment
Note: An alternative method to remove contaminating tRNA species is to switch from a glucose rich to glucose depleted media to promote ribosome runoff. However, this does change the metabolic status of the yeast cells, which could affect ribosome function and concentration.
In order to strip the ribosomes from endogenous peptidyl-tRNA, a treatment with puromycin is performed.
4. Purification of ribosomes by sedimentation through glycerol cushions
5. Representative results:
An example of RNA species extracted from each of the three major steps of the protocol is shown in Figure 2. While rRNAs are the major species present in total cell lysates (T) these also contain a large number of other RNA species. Ribosomes purified from the Sulfolink column (SL) also contain a large amount of tRNAs due to the high affinity of the column bed for these species as well. Treatment of this fraction with puromycin results in hydrolysis of peptides from peptidyl-tRNAs, and promotes dissociation of these species from ribosomes. The puromycin treated samples (Pm) lack co-purifying tRNA species, and thus represent completely pure ribosomes. As previously reported8, these ribosomes are highly intact and biochemically active, making them ideal substrates for detailed functional and structural analyses.
Figure 1. Method Flowchart. Chromatographic purification of ribosomes using the cysteine linked sulfolink resin is depicted.
Figure 2. Representative analysis of ribosome preparations. Total RNA species were extracted from total cell lysates (T), Sulfolink purified ribosomes (SL) and Sulfolink purified ribosomes subsequently treated with puromycin and sedimented through a glycerol cushion (Pm). Lane M represents RNA size markers. Bands representing 25S and 18S rRNAs, as well as tRNAs and other RNA species are indicated. All lanes were loaded with 2 μg of RNA.
Ribosome purification protocols basically involve lysing cells, harvesting a cytosolic fraction from a low speed spin, and then pelleting ribosomes by high speed centrifugation 1. While a few novel methods have been used for purifying bacterial ribosomes, the same had not been true for eukaryotes 2-4. Although additional steps have been added along the years, e.g. salt washes, and glycerol cushions (e.g. see 5), biochemical and structural studies of yeast ribosomes have been hampered by their tendency to become degraded by endogenous degrading enzymes during the purification process, most likely due to the long periods of time during the ultracentrifugation steps during which ribosomes are exposed to these classes of enzymes 6.
The major problem associated with traditional protocols is the co-purification of proteases and nucleases with ribosomes. This results in their degradation during the purification process, resulting in lower yields of biochemically active ribosomes. The high levels of proteases and nucleases present in clinical isolates of pathogenic bacteria led to the development of a chromatographic method for ribosome purification using a cysteine charged Sulfolink resin 7. The rRNAs and proteins derived from bacterial ribosomes isolated using this method showed much lower levels of degradation, and the ribosomes so purified were significantly more able to bind erythromycin and to synthesize proteins. The specific chemistry of ribosome binding to the Sulfolink resin is unknown, however it has been speculated that it involves hydrophobic interactions 7. These observations suggested that the cysteine charged Sulfolink resin chromatography method may also be applicable to yeast ribosomes, and if so, that it could solve many of the problems described above. Thus we adapted this protocol for isolation of intact, highly active yeast ribosomes. The column chromatographic method rapidly and efficiently results in separation of a significant fraction of contaminating nucleases and proteases from ribosomes resulting in purer, more biochemically active ribosome preparations with enhanced biochemical and structural properties, which scales well to higher quantities of ribosomes 8. No significant differences were seen on a denaturing protein gel between traditionally purified ribosomes and those purified via column chromatography, indicating that these ribosomes contain all the same ribosomal elements as traditionally purified ribosomes.
There are some steps in protocol that require special attention. With regard to disruption of yeast cells, a precise 1:1 ratio of cell suspension to glass beads (vol/vol) is critical. Typically, ˜80% of cells are disrupted in 2 min. Importantly, over-disruption should be avoided to prevent the release of degrading enzymes from cell organelles. Although ribosomes obtained directly from the Sulfolink column were shown to be nearly completely free of protease and nuclease contamination, the observation of high levels of tRNA in this fraction indicated that the resin binds tRNA as well 7,8. We have found that the presence of tRNA species interferes with downstream biochemical assays e.g. ribosome/ligand binding, assays of peptidyltransferase activity and rRNA structural analyses. Puromycin treatment 9 of chromatographically isolated ribosomes results in production of peptidyl-puromycin, which is released from ribosomes, along with deacylated tRNAs 10-14. The final overnight high speed centrifugation of samples through a glycerol cushion is critical for removing the remaining free tRNAs from the ribosome samples. If separate subunits are required, they may be obtained by sedimentation through a high salt sucrose gradient15
The authors have nothing to disclose.
We wish to thank all of the members of the Dinman laboratory, including Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy, and Michael Rhodin for their help and input on this project. This work was supported by grants to AM from the American Heart Association (AHA 0630163N) and to JDD from the National Institutes of Health (5R01 GM058859-12). JAL was supported by an American Reinvestment and Recovery Act of 2009 supplement to the parent grant (3R01GM058859-11S1).
Name of the reagent | Company | Catalogue number |
Sulfolink Coupling resin | Pierce | 20402 |
Mini bead-beater 16 | Biospec | 607 |
Optima Max E ultracentrifuge | Beckman Coulter | |
Legend RT | Sorvall | |
0.5 mm Glass beads | Biospec | 11079105 |
Tris | Sigma-Aldrich | 252859 |
EDTA | Sigma-Aldrich | E9884 |
DTT | Sigma-Aldrich | 43815 |
HEPES | Sigma-Aldrich | 54459 |
NH4Cl | Sigma-Aldrich | A9434 |
KCl | Sigma-Aldrich | 60128 |
Mg(OAc)2 | Sigma-Aldrich | M5661 |
KOH | Sigma-Aldrich | 221473 |
Glycerol | Sigma-Aldrich | G5516 |
Puromycin | Sigma-Aldrich | P7255 |
GTP | Fermentas | R0461 |