Translation and Ricin Castor beans and castor oil
Translation and Ricin
Castor beans and castor oil a. Ricinus communis - the castor plant b. The colorful fruit contains mottled castor beans (seeds) c. A greenish extract from the seeds, castor oil, has been used for many years to a. ease constipation, b. induce vomiting, c. a punishment for children (tastes HORRIBLE). d. Now it’s an ingredient in some plastics and for many other odd uses.
Castor bean poisoning a. Castor oil is not toxic (nauseating, but not toxic) b. What is left of the bean after oil extraction is the water-soluble portion, which contains the cytotoxic protein ricin. c. 5% of the dry weight of a castor bean is ricin! d. Symptoms: a. abdominal pain and vomiting b. diarrhea, sometimes bloody c. severe dehydration, d. a decrease in urine e. a decrease in blood pressure f. Death within 3 -4 days
Best known use of ricin… Assasination of Georgi Markov London, 1978 a. 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
Inept uses of ricin… Las Vegas Man Kills Self With Ricin a. 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.
Inept uses of ricin… Two women in plot to poison one's husband February 29, 2008 a. 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.
Mechanism of Toxicity a. How does ricin kill? b. Ricin kills cells by permanently disabling ribosomes, and therefore translation.
Genetic Code a. Consists of a triplet code a. That is, a sequence of three bases codes for a particular amino acid. b. There are 43, or 64 possible combinations c. A group of 3 bases coding for an amino acid in m. RNA is called a codon
Genetic Code a. Degenerate: an amino acid can be coded for by more than one codon b. Unambiguous: each condon indicates a single, specific amino acid c. Non-overlapping: when translated, the "reading frame" is advanced 3 bases at a time d. 61 codons are for amino acids, and the remaining three are "stop codons" that terminate the polypeptide
Stages of Translation
t. RNA Acceptor Stem Anticodon
Wobble a. There are NOT 61 t. RNAs b. Wobble: Base-pairing in the 3 rd position of the anticodon is not strict c. This lets multiple codons be recognized by one t. RNA Base in Anticodon Bases in 3 rd position of Codon A U C G U A or G G C or U I (inosine) U, C or A
Inosine a. Only found in t. RNA, and in the 3 rd position of the anticodon b. Inosine can base pair with U, C or A c. Example: Isoleucine (3 codons) a. Anticodon: UAI b. Codons: AUU, AUC, AUA
Aminoacyl-t. RNA Transferases a. Link amino acids to the correct t. RNA to make “activated” t. RNA b. One per amino acid c. Uses ATP to complete the ester bond d. The free energy of this bond is used to form the peptide bond during translation.
m. RNA a. Leader: Everything before the start codon b. Trailer: Everything after the stop codon c. Coding sequence: Everything from and including the start codon up until the stop codon
The bacterial ribosome a. 50 S and 30 S subunits in bacteria b. S stands for “Svedberg units” c. Four sites: a. m. RNA binding site b. A site (incoming t. RNA) c. P site (growing polypeptide) d. E site (discharged t. RNA)
Initiation a. Initiation factors (IF) b. Formyl-methionine (f. Met) c. Ribosome-binding site d. Energy from GTP bound to IF 2 is used to complete assembly
…in eukaryotes a. Methionine rather than f. Met b. Different initiation factors c. No ribosome binding site d. “Forward search” to find the start codon a. Complex of IF 2 -GTP and t. RNAMet binds to 5` cap b. Small subunit binds c. This complex searches forward to the first AUG d. The large subunit binds
Elongation a. Binding of t. RNA b. Peptide bond formation c. Translocation
Elongation
Simplified translation animation
Elongation in words… a. The P-site contains either methionine-t. RNA, or the growing peptide chain. A new AA-t. RNA arrives bound to EF-Tu (elongation factor Tu) with two bound GTPs. b. If the codon-anticodon match, hydrolysis of GTP is used to insert the new AA-t. RNA into the A site. a. b. The polypeptide chain is transferred to the AA in the A site. Peptidyl transferase is a ribozyme - one of the r. RNAs acts as an enzyme to catalyze the transfer. b. Energy for peptide bond formation is provided by the ester bond between the AA and t. RNA a. c. EF-G binds with one GTP a. Hydrolysis of GTP causes a shift of the ribosome along the m. RNA until the growing chain is back in the P site, and the empty t. RNA is in the E site.
Termination a. Terminates when stop codon arrives at the A site. b. A release factor binds c. The peptide chain transfers to a water instead of to an amino acid, creating a free carboxyl group d. The translational complex falls apart
Odds n’ ends… a. Folding proceeds as the peptide chain is synthesized. b. 1 ATP and 3 GTPs are used for each amino acid added. c. Many ribosomes proceed along a single m. RNA in a chain polyribosomes - to maximixe efficiency. d. Polycistronic m. RNA can contain multiple ribosome binding sequences - one for each protein in the operon - allowing simultaneous but separate translation of all encoded proteins.
Mutations a. Missense – a base change leads to a change of amino acid b. Nonsense – a base change leads to a stop codon c. Silent – a base change has no effect on the amino acid sequence
Mutations a. Frameshift – a base is inserted or deleted from the sequence, leading to a shift in the “reading frame” of the ribosome.
Self test a. What would the consequence be of each of these mutations, when considered in terms of protein function? • • Silent? Nonsense? Frameshift? Missense?
Ricin a. A dimeric protein b. Ricin A: A cytotoxin, but cannot get into cells efficiently c. Ricin B: A lectin that is not cytotoxic, but does enter cells efficiently. Also an agglutinin. d. Bound together by a disulfide bond
Ricin: cell entry a. Ricin A/B bind to cell surface sugars (glycoproteins and glycolipids) and are internalized. b. Vesicles are shuttled through the endosomes. c. Some ricin is returned to the cell surface… d. …more is degraded in the lysosome… e. … and still more is sent “back” to the trans-Golgi. 7 f. Again, some is returned to the cell surface g. Some escapes the Golgi into the cytoplasm 3 1 6 5 2 4
Ricin: cytotoxicity a. In the cytoplasm, ricin A and B separate. b. Q: What is it about the cytoplasmic environment that allows them to separate? c. Ricin A is an enzyme that depurinates a specific adenine on a r. RNA d. This inhibits the ability of the large ribosomal subunit to bind elongation factors e. Translation stops! f. As an enzyme, one ricin molecule can depurinate up to 50, 000 r. RNA molecules, thus inactivating the same number of ribosomes and killing the cell!
Applications a. For what might ricin be useful, besides a weapon? b. Research: studying the mechanisms of translation c. Research: studying protein binding to nucleic acids d. Cancer: if you can target even a single ricin molecule to a cancer cell, it’s dead!
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