National Center for Case Study Teaching in Science
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National Center for Case Study Teaching in Science Rice ’n Beans or Ricin Beans? A Deadly Swap Supplemental Presentation by Ann T. S. Taylor Department of Chemistry Wabash College, Crawfordsville, IN
http: //abcnews. go. com/GMA/video/ricin-letters -mailed-obama-elvis-impersonator-suspectreleased-19029161 2
Goals for today • Understand the types of experiments that can be used to monitor translation, including radionucleotide tracers, polyribosome isolation, and modification. • Apply and extend the concepts of translation to predict and explain inhibitor effects. • Form hypothesis and logical experimental techniques to explore translation. 3
100 90 % incorporation 80 70 60 3 H uridine 3 H thymidine 3 H leucine 50 40 30 20 10 0 0 1 5 10 �g of ricin/m. L of cell culture Figure 1. Incorporation of tritiated reagents in tumor cells (Lin et al. , 1971). 4
Group report out: Ways to inhibit translation 5
Components for an in vitro translation system 6
Phosphocreatine and creatine kinase 7
4. 5 [14 -C] Phe incorporated (counts/min 4 3. 5 3 2. 5 control 2 poly-U 1. 5 poly-U + ricin 1 0. 5 0 0 10 20 Time (minutes) 30 Figure 2. Effect of ricin on polyphenylalanine synthesis. Redrawn from Figure 2 of Olsnes and Phl, 1972. 8
How would you separate free 14 Cphenylalanine from poly-14 C-Phe? • Size exclusion chromatography • TCA precipitation 9
Polyribosome fractionation Unincorporated labeled amino acids 5% Sucrose Centrifuge 55% Sucrose 10
Figure 3. Ribosome sucrose gradients upon treatment with ricin or a translation inhibitor, ATA. Olsnes and Pihl, 1972. 11
Figure 4. Table 2 and caption in Sperti et al. , 1973. 12
Figure 5. Poly(U)-directedpolyphenylalanine synthesis catalysed by isolated control subunits and by isolated subunits treated with ricin. Detail of Panel A, Figure 3, in Endo et al. , 1987. 13
EF interaction changes % EF-ribosome binding compared to control Elongation factor binding after ricin treatment 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% EF-1 EF-2 Figure 6. Binding of radioactively labelled elongation factors to ribosomes. Based on Nilsson and Nygard, 1986. 14
32 P release in the presence of gamma-32 P GTP pmol 32 P released 150 control ricin 100 50 0 -Phe-t. RNA +Phe-t. RNA Figure 7. Release of 32 PO 43 - from ribosomes treated with 32 P-GTP and EF-1 in the presence and absence of Phe-t. RNA. Based on Nilsson and Nygard, 1986. 15
Ricin itself http: //www. proteopedia. org/wiki/index. php/Ricin 16
- National center for case study teaching in science
- National center for case study teaching in science
- National center for case study teaching in science
- National center for case study teaching in science
- National center for case study teaching in science
- National center for case study teaching in science
- National center for case study teaching in science
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