Translation and Ricin

1. Translation and Ricin

2. Castor beans and castor oil • Ricinus communis - the castor plant • The colorful fruit contains mottled castor beans (seeds) • A greenish extract from the seeds, castor oil, has been used for many years to • ease constipation, • induce vomiting, • a punishment for children (tastes HORRIBLE). • Now it’s an ingredient in some plastics and for many other odd uses.

3. Castor bean poisoning • Castor oil is not toxic (nauseating, but not toxic) • What is left of the bean after oil extraction is the water-soluble portion, which contains the cytotoxic protein ricin. • 5% of the dry weight of a castor bean is ricin! • Symptoms: • abdominal pain and vomiting • diarrhea, sometimes bloody • severe dehydration, • a decrease in urine • a decrease in blood pressure • Death within 3-4 days

4. Best known use of ricin… Assasination of Georgi Markov London, 1978 Bulgarian dissident is stabbed in the leg by a sharp tip on the end of an umbrella, depositing a small pellet containing ricin. He died four days later. The pellet contained only 200μg of ricin

5. Inept uses of ricin… Las Vegas Man Kills Self With Ricin POSTED: 8:39 a.m. EST March 3, 2003 LAS VEGAS -- Authorities in Las Vegas think a 60-year-old man used a homemade batch of the poison ricin to take his life. The Las Vegas Review-Journal says the man injected himself with ricin at his home late Friday, setting off a public health alert and prompting fears of bioterrorism.

6. Inept uses of ricin… Two women in plot to poison one's husband February 29, 2008 Las Vegas (CNN) -- man who stayed in a Las Vegas hotel room where ricin was found is in critical condition at a hospital, where he has been since mid-February... Firearms and an "anarchist type textbook" were found in the same motel room where several vials of ricin were found, police reported.

7. Mechanism of Toxicity • How does ricin kill? • Ricin kills cells by permanently disabling ribosomes, and therefore translation.

8. Genetic Code • Consists of a triplet code • That is, a sequence of three bases codes for a particular amino acid. • There are 43, or 64 possible combinations • A group of 3 bases coding for an amino acid in mRNA is called a codon

9. Genetic Code • Degenerate: an amino acid can be coded for by more than one codon • Unambiguous: each condon indicates a single, specific amino acid • Non-overlapping: when translated, the "reading frame" is advanced 3 bases at a time • 61 codons are for amino acids, and the remaining three are "stop codons" that terminate the polypeptide

10. Stages of Translation

11. AcceptorStem Anticodon tRNA

12. Wobble • There are NOT 61 tRNAs • Wobble: Base-pairing in the 3rd position of the anticodon is not strict • This lets multiple codons be recognized by one tRNA

13. Inosine • Only found in tRNA, and in the 3rd position of the anticodon • Inosine can base pair with U,C or A • Example: Isoleucine (3 codons) • Anticodon: UAI • Codons: AUU, AUC, AUA

14. Aminoacyl-tRNA Transferases • Link amino acids to the correct tRNA to make “activated” tRNA • One per amino acid • Uses ATP to complete the ester bond • The free energy of this bond is used to form the peptide bond during translation.

15. mRNA • Leader: Everything before the start codon • Trailer: Everything after the stop codon • Coding sequence: Everything from and including the start codon up until the stop codon

16. The bacterial ribosome • 50S and 30S subunits in bacteria • S stands for “Svedberg units” • Four sites: • mRNA binding site • A site (incoming tRNA) • P site (growing polypeptide) • E site (discharged tRNA)

17. Initiation • Initiation factors (IF) • Formyl-methionine (fMet) • Ribosome-binding site • Energy from GTP bound to IF2 is used to complete assembly

18. …in eukaryotes • Methionine rather than fMet • Different initiation factors • No ribosome binding site • “Forward search” to find the start codon • Complex of IF2-GTP and tRNAMet binds to 5` cap • Small subunit binds • This complex searches forward to the first AUG • The large subunit binds

19. Elongation • Binding of tRNA • Peptide bond formation • Translocation

20. Elongation

21. Simplified translation animation

22. Elongation in words… • The P-site contains either methionine-tRNA, or the growing peptide chain. • A new AA-tRNA arrives bound to EF-Tu (elongation factor Tu) with two bound GTPs. • If the codon-anticodon match, hydrolysis of GTP is used to insert the new AA-tRNA into the A site. • The polypeptide chain is transferred to the AA in the A site. • Peptidyl transferase is a ribozyme - one of the rRNAs acts as an enzyme to catalyze the transfer. • Energy for peptide bond formation is provided by the ester bond between the AA and tRNA • EF-G binds with one GTP • Hydrolysis of GTP causes a shift of the ribosome along the mRNA until the growing chain is back in the P site, and the empty tRNA is in the E site.

23. Termination • Terminates when stop codon arrives at the A site. • A release factor binds • The peptide chain transfers to a water instead of to an amino acid, creating a free carboxyl group • The translational complex falls apart

24. Odds n’ ends… • Folding proceeds as the peptide chain is synthesized. • 1 ATP and 3 GTPs are used for each amino acid added. • Many ribosomes proceed along a single mRNA in a chain - polyribosomes - to maximixe efficiency. • Polycistronic mRNA can contain multiple ribosome binding sequences - one for each protein in the operon - allowing simultaneous but separate translation of all encoded proteins.

25. Mutations • Missense – a base change leads to a change of amino acid • Nonsense – a base change leads to a stop codon • Silent – a base change has no effect on the amino acid sequence

26. Mutations • Frameshift – a base is inserted or deleted from the sequence, leading to a shift in the “reading frame” of the ribosome.

27. Self test • What would the consequence be of each of these mutations, when considered in terms of protein function? • Silent? • Nonsense? • Frameshift? • Missense?

28. Ricin • A dimeric protein • Ricin A: A cytotoxin, but cannot get into cells efficiently • Ricin B: A lectin that is not cytotoxic, but does enter cells efficiently. Also an agglutinin. • Bound together by a disulfide bond

29. Ricin: cell entry • Ricin A/B bind to cell surface sugars (glycoproteins and glycolipids) and are internalized. • Vesicles are shuttled through the endosomes. • Some ricin is returned to the cell surface… • …more is degraded in the lysosome… • … and still more is sent “back” to the trans-Golgi. • Again, some is returned to the cell surface • Some escapes the Golgi into the cytoplasm 3 1 2 6 5 7 4

30. Ricin: cytotoxicity • In the cytoplasm, ricin A and B separate. • Q: What is it about the cytoplasmic environment that allows them to separate? • Ricin A is an enzyme that depurinates a specific adenine on a rRNA • This inhibits the ability of the large ribosomal subunit to bind elongation factors • Translation stops! • As an enzyme, one ricin molecule can depurinate up to 50,000 rRNA molecules, thus inactivating the same number of ribosomes and killing the cell!

31. Applications • For what might ricin be useful, besides a weapon? • Research: studying the mechanisms of translation • Research: studying protein binding to nucleic acids • Cancer: if you can target even a single ricin molecule to a cancer cell, it’s dead!