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?

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

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

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

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

Group report out: Ways to inhibit translation 5

Components for an in vitro translation system 6

Components for an in vitro translation system 6

Phosphocreatine and creatine kinase 7

Phosphocreatine and creatine kinase 7

4. 5 [14 -C] Phe incorporated (counts/min 4 3. 5 3 2. 5 control

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

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

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.

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 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

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

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

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

Ricin itself http: //www. proteopedia. org/wiki/index. php/Ricin 16