1. Translation
Chapter 9

2. Overview Occurs on ribosomes-large aggregates of rRNA and protein
tRNA acts as amino acid carriers
Prokaryotes—occurs simultaneously with transcription and mRNA degradation
Eukaryotes—occurs in cytoplasm
mRNA translated 5’3’
Protein synthesis aminocarboxy

3. Protein Synthesis
Polymerization of amino acids: condensation reaction (dehydration synthesis)

4. ~Universal Genetic Code
Codons—sets of 3 nucleotides corresponding to a single amino acid
Each codon specifies a single amino acid
More than one codon can specify the same amino acid
code is said to be degenerate
Some aa correspond to a single codon
AUG—initiator codon, methionine (Met, M)
UGG–Tryoptophan (TrP, W)
Often codons encoding the same aa differ onl;y at the 3rd nucleotide
Codons—sets of 3 nucleotides corresponding to a single amino acid
Each codon specifies a single amino acid
More than one codon can specify the same amino acid
code is said to be degenerate
Some aa correspond to a single codon
AUG—initiator codon, methionine (Met, M)
UGG–Tryoptophan (TrP, W)
Often codons encoding the same aa differ onl;y at the 3rd nucleotide

5. ~Universal Genetic Code
The Genetic Code
Chart shows aa assignments
Non-random
Tend to be clustered
Reflects similar codons specifying the same aa
Spontaneous mutations causing a single base change
May not cause an aa change
Similar aa are specified by similar codons
Greatest similarities in first two nucleotides
eg glycine – 4 codons – all GGX
Greatest variability in third nucleotide of the triplet
QUIZ—Codon for R, K, V, F
aa for UCG, CCA, GGG

6. Why~Universal? Exceptions
GUG sometimes used as a start
Mammalian mitochondria
Ciliated protozoa
Selenocysteine
NH3+
COO-
H-C-CH2Se
GUG sometimes used as a start (bacteria)
Mammalian mitochondria- AUA start, other codons different than consensus. EX UGA-Trp (not Stop)
AGG, AGA-Stop (not Arg)
Ciliated protozoa—Ex UAA and UAG—Gln
Selenocysteine
An essential amino acid for selenoproteins
EX. Glutathione oxidase
Uses unique tRNA, initially bound to Ser
Anticodon recognizes UGA (Stop) as Sel
Signals in 3’ region determine Stop or Sel
Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

7. Selenocysteine The 21st amino acid?
An essential amino acid for selenoproteins EX. Glutathione oxidase
Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt).
Anticodon recognizes UGA (Stop) as Sec
Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec
Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu)
The 21st amino acid?
An essential amino acid for selenoproteins EX. Glutathione oxidase
Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt).
Anticodon recognizes UGA (Stop) as Sec
Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec
Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu)
Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

8. Degeneracy—Wobble Hypothesis
Explains how some tRNA recognize more than one codons
tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide
This third loose base pairing interaction is called wobble
Note: only certain bases can substitute for others
Explains how some tRNA recognize more than one codons
tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide
This third loose base pairing interaction is called wobble
Note: only certain bases can substitute for others
How?
Canonical base pairing with the 1st two codon bases
Loose, weak base pair interactions with the 3rd codon base
#1 nucleotide of the anticodon is in a flexible domain of the tRNA
Why?
Kinetic advantage
tRNA can dissociate more readily from the RNA template
Allows faster protein synthesis

9. Wobble Example This UCA codon was read by the
tRNA with a UGA anticodon
But if this UCA was UCG, it would still have been read by the tRNA with a UGA anticodon
In DNA , no pyr-pyr or pur-pur, but RNA is single stranded, allows more flexibility.
Proposed by Crick (model building). Confirmed by experimentation.
Implications for GE? Primer design

10. Codon Usage
More than one codon exists for most amino acids (except Met and Trp)
Organism may have a preferred codon for a particular amino acid
Codon usage correlates with abundance of tRNAs (preferred codons are represented by abundant tRNAs)
Rare tRNAs correspond to rarely used codons
mRNAs containing rare codons experience slow translation
Implications for GE?

11. Amino Acyl Synthetase and tRNA
Amino acyl synthetases catalyze attachment of aa to its appropriate tRNA
One for each amino acid
tRNA
Derived from large 1º transcript
Heavily modified, unusual bases
Extensive folding due to internal H-bonding
There are 20 amino acids and many tRNAs--over 400 possible combinations between the two. Redundancy in the code means that there are more codons than amino acids.
It is the process of linking the appropriate tRNA to its specific amino acid that is considered the second genetic code.
The specificity of the reaction is governed by tRNA identity elements that are recognized by enzymes called tRNA synthetases.

12. Amino Acyl Synthetase Carboxy end of aa attached to -phospate of ATP
AMP released as carboxy end of amoinoacyl group transferred to O at C-3 of 3’nt
When aa is attached, tRNA is charged or acylated
No aa = uncharged
Wrong aa = mischarged
NOTE PPi
Carboxy end of aa attached to -phospate of ATP
AMP released as carboxy end of aa transferred to O at C-3 of 3’nt
When aa is attached, tRNA is charged or acylated
No aa = uncharged
Wrong aa = mischarged
NOTE PPi

13. tRNA Activation by Aminoacyl tRNA Synthetases
1. Aminoacyl-AMP formation: O HO
(-)O R P O O
O(-) P O O
Adenine +H 3 N C P +
PPi R O O O O O-
Adenine O
O(-) P +H 3 N C O O O- O OH OH
Aminoacyl adenylate
(Aminoacyl-AMP)
2Pi OH OH
2. Aminoacyl transfer to the appropriate tRNA:
The generation of aminoacyl-tRNA's is a two-step process carried out by a family of enzymes called aminoacyl-tRNA synthetases.
1.The amino acid is first activated by conversion of the free carboxylic acid to aminoacyl adenlylate. Although stable, the aminoacyl-AMP intermediate does not leave the enzyme.
2. The amino acid is then transferred to the appropriate tRNA
What happens to PPi?
Because of the pyrophosphatases present in the cell, the pyrophosphate produce (Ppi, inorganic pyrophosphate), is quickly hydrolyzed to Pi, rendering the reaction thermodynamically favorable and irreverisble R R O O O
Adenine +H 3 N C P +
HO-ACC-tRNA +H 3 N C
ACC-tRNA +
AMP O O O- O O OH OH
Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi

14. tRNA Function and Structure
Anticodon-complementary to codon on mRNA
Amino attachment (CCA) site
Other recognition sites
DHU loop
TC Loop
Extra arm (variable)
NOTE: also unusual bases observed acceptor stem
acceptor stem
Anticodon-complementary to codon on mRNA. NOTE: no normal tRNA complementary to UGA, UAA, or UAG (except tRNSSec)
Amino attachment (CCA) the 3’ end of the tRNA. This is the place at which the specific amino acid is attached
These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G·U wobble base pair located at the third position within the acceptor helix of alanine tRNA.

15. tRNA Recognition by Amino Acyl Synthetase
Sequence elements in each tRNA are recognized by its specific synthetase including:
One or more bases in acceptor stem
Base at position 73 “Discriminator base”
Seems to play a major role in many cases, but in other cases it is completely ignored.
In many, at least one anticodon base
The features that permit recognition are not universal, yet there are some general rules. Recognition is not limited to the anticodon end of the tRNA.
Discriminator base is invariant in every tRNA for a particular amino acid

16. No common set of rules for tRNA recognition !!!
Recognition (cont’d)
No common set of rules for tRNA recognition !!!
Anticodon region is not the only recognition site
The "inside of the L" and other regions of the tRNA molecule are also important
Specificity of several aminoacyl-tRNA synthetases determined by:
one or more bases in anticodon
one or more bases in the acceptor stem
discriminator base 73

17. Mischarging
Observation: several aa similar in size and shape, but mischarging rare.
Editing carried out by aminoacyl tRNA synthetase
Ex Double sieve of isoleucine synthetase
Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit.
Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed).
Sites can also distinguish based on hydrophobicity
There are 20 amino acids and many tRNAs--over 400 possible combinations between the two. Redundancy in the code means that there are more codons than amino acids.
It is the process of linking the appropriate tRNA to its specific amino acid that is considered the second genetic code.
The specificity of the reaction is governed by tRNA identity elements that are recognized by enzymes called tRNA synthetases.
Observation: several aa similar in size and shape, but mischarging rare.
Editing carried out by aa tRNA synthetase
Ex Double sieve of isoleucine synthetase
Expect 1 mischarge/225 events. Consequence? 1 misincorporated aa every time Ile is indicated. Intolerable.
Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit.
Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed).
Sites can also distinguish based on hydrophobicity

18. Isoleucil-tRNA Synthetase: Proofreading Based on Size
Recognition of aa by tRNA synthetases is based on size and hydrophilicity of aa chainThe recognition of amino acids by aminoacyl-tRNA synthetases is generally based on the size of the amino acid side chain.
 •Acylation site rejects amino acids that are larger than the correct one because the binding site is too small.
•Hydrolytic site destroys activated intermediates that are smaller than the right amino acid.
Example: Valine vs isoleucine (isoleucine has an extra methyl group)
If valine is mistakenly activated by tRNA coding for isoleucine, it is hydrolyzed preventing its incorporation in tRNAIle :
Acylation site rejects aa that are too large to fit in active site. Hydrolytric site destroys activated intermedeated that are smaller than correct aa
Many synthases possses hydrolytic site. If Val is mistakenly activated the Ile tRNA, it will be hydrolyzed by smaller hydrolytic site
Larger
Acylation Site
Smaller
Hydrolytic Site
Larger
Acylation Site
Smaller
Hydrolytic Site CH 3 CH 3 H 3 C CH 3 O O +H 3 N NH 3 + O
tRNAIle O
tRNAIle CH 3 H 3 C CH 3 CH 3 O O +H 3 N +H 3 N O
tRNAIle O
tRNAIle
Ile
Val
Misacylation
Correct Acylation

19. Valyl tRNAVal Synthetase Proofreading: Hydrophobic/Polar Recognition Motif
Acylation and hydrolytic sites can also discriminate based on hydrophobic verses polar interactions.
Example: Valine vs threonine (difference is –OH in place of CH3)
If threonine is mistakenly activated by tRNA coding for valine, it is hydrolyzed preventing its incorporation in tRNAVal :
 Anticodon does not always participate in recognition
Mutational studies have shown that recognition features are relatively simple
Ale – GC pair in tRNA acceptor stem
Aminoacyl-tRNA Synthetases Have two roles
Aminoacyl-tRNA synthetases do the critical job - linking the right amino acid with "cognate" tRNA
They act as a “scaffold” to match up the tRNA with its correct (“cognate”) amino acid
They catalyze a two-step reaction
This generates an ester linkage between
-the 3’OH of the tRNA (on the acceptor stem)
-and the COO- group of the amino acid2) This reaction activates the amino acid for protein synthesisAll members of one set of tRNAs for a particular amino acid (isoacceptor tRNAs) are served by one Aa tRNA synthetase.
Hydrophobic
Acylation Site
Polar
Hydrolytic Site
Hydrophobic
Acylation Site
Polar
Hydrolytic Site 3 HC CH 3 H 3 C OH O O +H 3 N NH 3 + O
tRNAVal
tRNAVal O
Difference in Hydrophobicity
CH3 CH 3 HO CH 3 O O +H 3 N +H 3 N O
tRNAVal O
tRNAVal
Val
Thr
Correct Acylation
Misacylation

20. Experiment (1962) tRNA-ACA Protein has Cys Cys-tRNA-ACA
Anticodon (recognizes UGU codon, encodes Cys)
tRNA-ACA
Cell-free extract amino acids & enymes
tRNA is charged with Cys
Protein has Cys
RNA template
UGUGUGUGUG...
Cys-tRNA-ACA
Treat w metal catalyst removes thiol groups
Charged amino acid is changed chemically
This experiment indicates the amino acid makes no contribution to accurate translation
Protein has Ala
RNA template
UGUGUGUGUG...
Ala-tRNA-ACA
Once an aminoacyl-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.

21. Protein Synthesis-3 Stages
Initiation
Elongation
Termination

22. Ribosomes Composition of eukaryotic and prokaryotic ribosomes
subunits, each composed of about 60% RNA and about 40% protein (by mass).
Small ribosomal subunit
16S rRNA (bacteria), 18S rRNA (eukaryotes)
21 (bacteria) to 33 (eukaryote) proteins
Initial binding of mRNA and initiator met-tRNA
Large ribosomal subunit
23S & 5S rRNA (bacteria), 28S, 5.8S, & 5S rRNA (eukaryotes)
31 (bacteria) to 49 (eukaryote) proteins
Forms complete, catalytic ribosome.
Mol. Biol. Gene, Fig

23. Composition of the E. coli Ribosome
50S subunit 23S & 5S RNA + 34 proteins
30S subunit 16S RNA + 21 proteins

24. Gross anatomy of the E. coli ribosome.
head
platform
stalk
Central protuberance
platform
ridge
stalk
Fig. 19.5

25. Initiation
In both prokaryotes and eukaryotes, protein synthesis begins with a specific initiating tRNA
In prokaryotes, initiator methionine amino group is methylated
Attached to special tRNA (fMet-tRNA)
Transformylase—adds formyl group
Deformylase—removes formyl group from Met of completed peptide
Formylation does not occur in eukaryotes

26. } Steps in Initiation Association of 30S subunit with
mRNA
fMet-tRNA
Initiation factors (3 proteins)
GTP
QUESTION: AUG encodes fMet and Met. How does 30S ribosome “know” which aa is to be inserted? }
30S Pre-initiation Complex

27. Initiator Codon Recognition
fMet-tRNA responds only to initiator codons (AUG, GUG, UUG [rarely])
Met-tRNA responds only to internal AUG
Meaning of codons dependent on their context, i.e., sequences nearby
In Eukaryotes: 5’ cap involvement
In Prokaryotes: Shine-Dalgarno Sequence
mRNA- (5’)AGGAG (3’)
16S rRNA- (3’)UCCUC(5’)
fMet-tRNA responds only to initiator codons (AUG, GUG, UUG [rarely])
Met-tRNA responds only to internal AUG
Meaning of codons dependent on their context, i.e., sequences nearby
In Euks: 5’ cap involvement
In Proks: Shine-Dalgarno Sequence
mRNA- (5’)AGGAGG(3’)
16S rRNA- (3’)UCCUCC(5’)

28. Shine-Dalgarno Interaction
Upstream from initiator AUG
Complementary to a stretch on 16S rRNA
Seen in virtually all prokaryotic mRNA

29. Initiation Factors in Protein Synthesis
IF-1
Promotes dissociation of ribosome.
IF-1 also blocks the A site of the small ribosomal subunit
 insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation.
IF-2
small GTP-binding protein (a GTPase).
Interacts with
Small subunit
IF1
fMet-tRNAfMet
IF-1
Promotes dissociation of ribosome.
IF-1 also blocks the A site of the small ribosomal subunit
 insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation.
IF-2
small GTP-binding protein (a GTPase).
Interacts with:
Small subunit
IF1
fMet-tRNAfMet

30. Initiation Factors in Protein Synthesis
IF-2 (cont’d)
IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit
IF-3
Binds small subunit, prevents reassociation w/ large subunit
binds mRNA to the 30S ribosomal subunit
frees it from its complex with the 50S subunit.
IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit
IF-3
Binds small subunit, prevents reassociation w/ large subunit
binds mRNA to the 30S ribosomal subunit
frees it from its complex with the 50S subunit.
As mRNA binds, IF-3 helps to correctly position the complex such that the tRNAfMet interacts via base pairing with the mRNA initiation codon (AUG).

31. 30S Pre-initiation Complex
IF-1
Promotes dissociation of ribosome.
IF-1 also blocks the A site of the small ribosomal subunit
 insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation.
IF-2
small GTP-binding protein (a GTPase).
Interacts with
Small subunit
IF1
fMet-tRNAfMet
IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit
IF-3
Binds small subunit, prevents reassociation w/ large subunit
binds mRNA to the 30S ribosomal subunit
frees it from its complex with the 50S subunit.

32. 70S Initiation Complex Assembly
30S pre-initiation= 30S subunit,IF1-3, mRNA, GTP, fMet-tRNAfMet
When fMet-tRNAfMet pairs with initiator codon, small subunit undergoes conformational change
Result: Release of IF-3
Large subunit can bind small subunit complex
Binding of large subunit stimulates GTPAse activity of IF-2/GTP
hydrolysis of GTP to GDP
IF-2/GDP and IF-1 fall off
RESULT: 70S ribosome with fMet-tRNAfMet in P-site of ribosome
As the large ribosomal subunit joins the complex,
GTP on IF-2 is hydrolyzed, leading to dissociation of IF-2-GDP and dissociation of IF-1.
A domain of the large ribosomal subunit serves as GAP (GTPase activating protein) for IF-2.
Once the two ribosomal subunits come together, the mRNA is threaded through a curved channel that wraps around the "neck" region of the small subunit..

33. RESULT: 70S Initiation Complex

34. Overview Dissociation of inactive 70S IF-1, IF-3 IF-2/tRNA, mRNA
fMet-tRNAfMet/initiator codon-releases IF-3
GTP Hydrolysis, releases IF-1, IF-2
Complete 70S complex

35. 70S Ribosome A (aminoacyl) site Small subunit Transfer RNAs
P (peptidyl) site
Large subunit
(50 S)
5’ end
Messenger RNA

36. Alternative View
A=Aminoacyl site
P=peptidyl site
E=Exit site

37. Elongation Overview
Aminoacyl tRNA complementary to codon in A-site moves into A-site
N-formyl-Met transferred from tRNAfMet to aminoacyl-tRNA in A-site
now have a dipeptide in the A-site
tRNAfMet leaves P-site
Ribosome moves along mRNA (translocation)
dipeptide in P-site
New aminoacyl-tRNA moves into A site, etc
INSERT

38. ELONGATION INSERT Elongation CHAIN ELONGATION:
3-stage elongation rxn cycle
Rate Up to 40 residues/ sec
Requires non-ribosomal proteins (elongation factors) and a ribozyme (part of 23S rRNA)
INSERT

39. Transpeptidation Reaction
Carboxy end of nascent peptide
Nucleophilic attack
A ring nitrogen (N) of the catalytic adenosine (at the CCA end of tRNA) may promote the reaction by extracting a H+ from the attacking amino N.
This H+ is then donated to the hydroxyl of the tRNA in the P site, as the ester linkage is cleaved.
Amino terminus of incoming amino acid

40. Transpeptidation Completed
The nascent polypeptide, one residue longer, is now linked to the tRNA in the A site.
However, translocation has already partly occurred, because peptide bond formation is associated with rotation of the single-stranded 3' end of the A-site tRNA toward the P-site, positioning the aminoacyl moiety for catalysis.
This rotary movement also positions the nascent polypeptide to feed into the entrance to the protein exit tunnel, which is located midway between A & P sites.

41. EF-G-GTP Role in translocation Binding site uncovered
EF-G-GTP occupies
Hydrolysis
GDP leaves
open A-site
Role in translocation.
After translocation, shift in A-site tRNA uncovers a binding site for EF-g-GTP in large subunit portion of A-site.

42. Translation Termination
“Stop” Codon
No anticodons, but are release factors
Proteins
Occupy A-site
Activate hydrolysis of peptide from peptidyl-tRNA

43. Translation Termination 2
Release factors
RF-1 and RF-2 recognize stop codons
RF-3 –stimulates dissociation of 70S ribosome after release of polypeptide chain
Anticodon recognition determined 3 aa
Release factors
RF-1 and RF-2 recognize stop codons
RF-I UAG
RF-2 UGA
Both --UAA
RF-3 –stimulates dissociation of from ribosome after release of polypeptide chain
Anticodon recognition determined 3 aa in RF-3 (a peptide anticodon).

44. Proofreading in Translation
Codon:anticodon base pairing
16S rRNA forms H-bonds with minor groove of codon:anticodon duplex only when correctly paired

45. 3. Proofreading in Translation
Correct base pairing allows EF-Tu bound to aa-tRNA to interact with factor binding center, inducing GTP hydrolysis and EF-Tu release
Incorrect base pairing
FBC not contacted
allows more time for EF-Tu GTP release
Correct base pairing allows EF-Tu bound to aa-tRNA to interact with factor binding center, inducing GTP hydrolysis and EF-Tu release
Incorrect base pairing allows more time for EF-Tu GTP release
Pausing caused by activation allows non-cognate tRNA to dissociate. It leaves before EF-Tu
not incorporated.

46. 4. Proofreading in Translation
Incorrectly paired tRNA can’t rotate into position for peptide bond formation
“tRNA accommodation”
Only correctly base-paired aa-tRNA remain associated w/ribosome as they rotate into the correct position for peptide bond formation. Rotation referred to as tRNA accomodation.

47. Antibiotics Translation the target of many antibiotics. EX
Site of Nucleophilic attack
Absent terminates translation
Puromycin

48. Eukaryotic Translation Factors
designated with the prefix "e"
EF-Ts replaced by
eEF-1
eEF-2 (target for diphtheria toxin)
Eukaryotic Translation Factors
designated with the prefix "e"
EF-Ts replaced by
eEF-1
eEF-2
NOTE: eEF-2 is a specific target for diphtheria toxin (catalyzes addition of ADP to eEF-2)
Toxin acts catalytically
Eventually kills cell by halting protein synthesis
a few µg sufficient to kill an unimmunized individual
Formerly a leading cause of childhood death
EF2 is only known substrate for diphtheria toxin
EF2 contains rare modification of one of histidine residues and this is site recognized by toxin
Mutant cells that cannot modify site are resistant
Addition of ADP-ribose inactivates EF2
Kills cells by irreversible block of protein synthesis
COMPOSED OF 2 PARTS:
B-fragment—binds receptor
A-fragment—business end, inserted by B-fragment
Therapeutic potential

49. Diphtheria Toxin
EF2 is only known substrate for diphtheria toxin
EF2 contains rare modification of one of histidine residues and this is site recognized by toxin
Mutant cells that cannot modify site are resistant
Addition of ADP-ribose inactivates EF2
Kills cells by irreversible block of protein synthesis
P. aeruginosa exotoxin A works same as diphtheria toxin