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. tRNA
Acceptor Stem
Anticodon

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
Base in Anticodon
Bases in 3rd position of Codon A U C G U
A or G G
C or U
I (inosine)
U, C or A

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. Binding of tRNA Peptide bond formation Translocation
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!