Identifying sites of protein synthesis in Chlamydomonas using erythromycin and cyclohexamide as protein synthesis inhibitors.
October 16, 2009
In living cells, prokaryotic or eukaryotic, the synthesis (construction) of proteins is accomplished by similar machinery. Amino acids, ribosomes, messenger RNA (mRNA), and transfer RNA (tRNA), are all necessary for the building of functional proteins in a cell. Ribosomes are the site of protein synthesis in a cell, and there are two different types, depending on the type of cell. Only the 70S ribosomes are found in prokaryotes (bacteria, archaea). Eukaryotes, (cells that contain a membrane-bound nucleus), have both 70S and 80S ribosomes. The 70S ribosomes are present in mitochondria and chloroplasts of eukaryotic cells (Willey, et al, 20083). The theory of endosymbiosis is based on the thought that mitochondria and chloroplasts were, at one time, prokaryotic organisms that were engulfed into a eukaryotic cell and formed an equally beneficial relationship. Since mitochondria and chloroplasts contain their own DNA and self-replicate, their genetic codes are passed along with the rest of the cell when reproduction occurs. The two types of ribosomes made in a eukaryotic cell will respond differently when given certain antibiotics (Nelson, et al 2009). This experiment focused on two: erythromycin and cyclohexamide. When given erythromycin, tRNA is inhibited from transferring from one site of ribosomal RNA (rRNA) to another site during protein synthesis. However, this inhibition only occurs in the 70S ribosome (DrugBank, 2011). In contrast, when a cell is given cyclohexamide, the 80S ribosomes are blocked from completing the process of elongation (the addition of triplet codons that are complimentary to an mRNA molecule). This occurs in the last step of elongation, during the translocation phase (Willey, et al, 20081). Translocation is the movement of the tRNA molecule from one part of the ribosome to another as it adds its complimentary amino acid opposite the mRNA strand. (Willey, et al, 20082). As a result of the inhibition actions, a cell given erythromycin will only produce proteins using 80S ribosomes (cytoplasmic proteins). Conversely, when given cyclohexamide, proteins synthesis of cytoplasmic proteins decreases (Dzyubinskaya, 2006).
In order to determine the number of proteins made by Chlamydomonas when treated with these antibiotics, the cells were treated with 35S, a radioactive isotope of sulfur. The algal cells used the sulfur in the synthesis of new proteins. It is then possible to measure the amount of radioactivity in the new proteins after the antibiotic treatment with a scintillation counter. As Chlamydomonas contains only a single chloroplast, which accounts for a significant amount of its volume, affecting protein synthesis in either type of ribosome will constitute a noticeable decrease in production. This experiment sets out to determine where the resultant proteins would have remained chloroplast and mitochondrial proteins, in the cytoplasm of the cell, or in the cell membrane.
Materials and Methods:
Into each of three microfuge tubes, one milliliter (1mL) of Chlamydomonas culture was inserted and spun at top speed (14,000 rcf) for two minutes. When completed, the supernatant was removed and discarded. Each of the three tubes was then resuspended with 50 microliters (µL) of erythromycin (100µg/mL), 50µL of cyclohexamide (10µg/mL), or 50µL of TRIS buffer (as the control).
Each of the three tubes was then mixed with 50µL of the radioactive sulfur isotope 35S and incubated at room temperature for 30 minutes. The samples were then centrifuged at top speed for five minutes, and the supernatant discarded. The remaining pellet was frozen at -20°C for 2 minutes, then thawed (room temperature for 2 minutes) 3 times to burst the cells. The cells were again resuspended, using 100µL of TRIS buffer and mixed....
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