Precipitation of peptidyl-tRNA by cetyltrimethylammonium bromide affords an extremely convenient and accurate means of following the reaction of nascent polypeptides with puromycin. Using this assay, I have investigated the modes of action of several antibiotics which inhibit polypeptide chain elongation. The polypeptide, alpha sarcin, has been shown to inhibit the binding of aminoacyl-tRNA to the ribosomal 'A' site as have the antibiotics, chartreusin and emetine. Additionally, it has been confirmed that sparsomycin and trichodermin inhibit the peptidyl transferase whereas cycloheximide probably inhibits translocation. The technique for measuring peptidyl-tRNA concentrations has also been used to detect inhibitors of initiation of protein synthesis. By this and other techniques, homoharringtonine has been shown to inhibit a step in initiation - possibly the same step as that inhibited by the chemically-unrelated antibiotic T-2 toxin. Cell-free extracts of S. cerevisiae and M. verrucaria have been prepared which are very active in poly U-directed protein synthesis. These extracts have been used to study resistance to the 12,13 epoxytrichothecene group of toxins either in a yeast mutant, strain TR1, or in the organism M. verrucaria which produces several 12,13 epoxytrichothecenes. Both strain TR1 and M. verrucaria are resistant to T-2 toxin in vitro because of features of their 60S ribosomal subunits. The effect of the 12,13 epoxytrichothecene, trichodermin, on poly U-directed protein synthesis by yeast cell-free extracts was also investigated. It was found that there were apparently two types of synthesis - one sensitive and one insensitive to trichodermin. The ratio of the two types of synthesis appeared independent of the manner in which the ribosomes were prepared but was influenced by ion concentrations and the mode of preparation of the high-speed supernatant fraction (S100). Both types of poly U-directed protein synthesis involved production of polyphenylalanine rather than peptidy1-polyphenylalanine. Cell-free extracts capable of poly U-directed protein synthesis were also produced from strains of the 12,13 epoxytrichothecene-producing organisms F. poae, F. sporotrichiodes and F. equiseti and the fungi, A. giganteus, which produces alpha sarcin. All the organisms were sensitive to their products in vitro but resistant in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:459376 |
| Date | January 1978 |
| Creators | Hobden, Adrian N. |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35254 |
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