4: Genome evolution
Exon Shuffling
3 types of exon shufflingexon duplication = the duplication of one or more exons within a gene (internal duplication)
exon insertion = exchange of domains between genes or insertions into a gene
exon deletion = the removal of a segment from a gene.
Mosaic Mosaic (or (or chimericchimeric)) protein protein = a protein encoded = a protein encoded by a gene that contains by a gene that contains regions also found in other regions also found in other genes. The existence of such genes. The existence of such proteins provides proteins provides evidenceevidence of of exon shuffling. exon shuffling.
exon shufflingexon shuffling
mosaic proteinsmosaic proteins
4: Genome evolution
Blood Clotting
Clotting - History
1863: Joseph Lister showed that blood is a fluid inside an ox, and undergoes clotting when put on a test glass.
Clotting – The end reaction
thrombinfibrinogen fibrin
After the wound is healed…
plasmin
thrombinfibrinogen fibrin
Clotting
no fibrin (peptides)
After the wound is healed…
plasminogen
Plasmin should only be activated when the wound is healed. Plasmin is created from plasminogen by an enzyme called plasminogen activator (TPA).
Tissue type plasminogen activator (TPA)
fibrin no fibrin
plasmin
Heart attacks
plasminogen
If TPA is given 1h after a heart attack it significantly increases the chance of surviving. (If you run out of TPA chewing aspirin will also do)
Tissue type plasminogen activator (TPA)
fibrin no fibrin
plasmin
After the wound is healed…
plasminogen
Urokinase catalyzes the same reaction as TPA.
urokinase
fibrin no fibrin
plasmin
tissue type plasminogen activator (TPA)
After the wound is healed…
plasminogen
prourokinase
urokinase
fibrin no fibrin
plasmin
tissue type plasminogen activator (TPA)
Prourokinase is the precursor of urokinase
After the wound is healed…
Prourokinase and TPA are very similar. They both catalyze the same reaction (prourokinase only after it is cleaved to urokinase). But, the difference is that TPA interacts with fibrin and urokinase – does not.
plasminogen
prourokinase
urokinase
fibrin no fibrin
plasmin
tissue type plasminogen activator (TPA)
Prourokinase and TPA – the domains
The difference is that TPA has another domain, the F1 domain (43 amino acids) that is missing in prourokinase.F1 = fibronectine type 1 module.F1 is responsible for the affinity of TPA to fibrin.
KR ProteaseEG KR
KR ProteaseF1 EG KR
Prourokinase
TPA
Prourokinase and TPA – the domains
What is the origin of the F1 domain?
KR ProteaseEG KR
KR ProteaseF1 EG KR
Prourokinase
TPA
Prourokinase and TPA – the domains
It probably came from another protein, called fibronectin.
F5 F3F2
F2 = Collagen binding domainF3 = Heparin biding domainF4 = Cell binding domain
2 5
F1
6
Six repeats of F1
F1
3
F3
9
F4 F3F1
3
S-S
S-S
F5 F3F2
2 5
F1
6
F1
3
F3
9
F4 F3F1
3
Fibronectin
Fibronectin can connect (F4) fibroblasts to fibrin (F1) to repair site of injury.
F5 F3F2
F2 = Collagen binding domainF3 = Heparin biding domainF4 = Cell binding domain
2 5
F1
6
Six repeats of F1
F1
3
F3
9
F4 F3F1
3
S-S
S-S
F5 F3F2
2 5
F1
6
F1
3
F3
9
F4 F3F1
3
Much more complicated
In fact, there are other domains in the clotting system that resemble each other. It looks like a big complicated puzzle of domain shuffling.
AP = apple module; EG = epidermal growth-factor; F1 & F2 = fibronectin type-1 & type-2; GA = -carboxy-glutamate domain; KR = kringle
TPA acquired its exons from other genes…
There are also many computer programs that analyze a given sequence, and search for homology in known existing domains.
4: Genome evolution
Phase limitations on exon shuffling
The phase of an intron
ATGGGATTCGTTAGCCATTT
Exon
Intron of phase 0: lies between two codons
Exon
The phase of an intron
Exon
Intron of phase 1: lies between the first and second positions of a codon
Exon
ATGGGATGTTAGTCCCATTT
The phase of an intron
Exon
Intron of phase 2: lies between the second and third positions of a codon
Exon
ATGGGATTGTTAGCCCATTT
The class of an exon
Exon of class 0-0. Starts at the beginning of a codon, and ends at the end of a codon.
GTTAGCCATTTGTT
The class of an exon
Exon of class 0-1. Starts at the beginning of a codon, and ends between positions 1 and 2 of a codon.
GTTAGCCATTTGTT
The class of an exon
Exon of class 2-1. Starts between positions 2 and 3 of a codon and ends between positions 1 and 2 of a codon.
GTTAGCCATTTGTT
Exon of class?
Symmetrical exons.
GTTAGCCATTTGTTT
Symmetrical exons are those that are multiples of 3 nucleotides?
(Otherwise, they are asymmetrical).
Tandem duplication of symmetrical exons
GTTAGCCATTTGTGCCATTTGTTT
Tandem duplication of symmetrical exons will not cause a frameshift mutation.
GTTAGCCATTTGTTT
Deletion of symmetrical exons
Deletion of symmetrical exons will not cause a frameshift mutation.
GCGTTAGCCATTTGTTTATTT
What about exons insertion?
Only symmetrical exons can be inserted without causing a frameshift mutation.
GTTAGCCATTTGTTTACCGATTTCAC
What about exons insertion?
But not all symmetrical exons can enter. For example, a 0-0 exon will cause a frameshift if
entered.
GTTAGCCATTTGTTTAGGTACGCCGATTTCAC
GGTACG
GTTAGCCATTTGTTTACCGATTTCAC
What about exons insertion?
0-0 exons can only be inserted in phase 0 introns1-1 exons can only be inserted in phase 1 introns2-2 exons can only be inserted in phase 2 introns
GTTAGCCATTTGTTTAGGTACGCCGATTTCAC
GGTACG
GTTAGCCATTTGTTTACCGATTTCAC
Prourokinase and TPA – exon classes
All recruited exons are of class 1-1. It might be a “frozen accident”: if the first one was 1-1, all the rest should also be 1-1…
KR ProteaseEG KR
KR ProteaseF1 EG KR
Prourokinase
TPA
4: Genome evolution
exonization and exon lost
Splicing
In splicing, introns are removed. There are signals in the DNA (in the mRNA) that direct the excision of introns.
exon1 exon2protein
exon1 exon2
exon1 exon2
mRNA
mature mRNA
exon1 exon2DNA
ExonizationMutations in the DNA that encode signals for intron excision might result in exonization of the intron.
protein
mRNA
mature mRNA exon1
DNA exon1
exon1
exon1
exon1 exon2
mutation in the splicing signal
Exon lostOf course, in a similar vain, exons can also be removed due to such mutations.
protein
mRNA
mature mRNA
DNA
exon1
exon2
mutation in the splicing signal
exon1
exon1
exon1
exon1
4: Genome evolution
Principal biochemical reactions in the synthesis of Principal biochemical reactions in the synthesis of fatty acidsfatty acids from from malonyl CoAmalonyl CoA in in eukaryotes and eubacteriaeukaryotes and eubacteria_____________________________________________________________________________________________________________________
Reaction Enzyme_____________________________________________________________________________________________________________________1. acetyl CoA + condensing-enzyme domain acetyl-condensing enzyme acetyl transferase2. malonyl CoA + acyl-carrier peptide malonyl-acyl-carrier peptide malonyl transferase3. acetyl-condensing enzyme + malonyl-acyl-carrier peptide -ketoacyl-carrier peptide -ketoacyl synthase-keto-acyl carrier peptide + NADPH + H+ -hydroxyacyl-carrier peptide + NADP+ -ketoacyl reuctase5. -hydroxyacyl-carrier peptide 2-butenoyl-acyl-carrier peptide + H2O -hydroxyacyl
dehydratase6. 2-butenoyl-acyl-carrier peptide + NADPH + H+ butyryl-acyl-carrier peptide + NADP+ enoyl reductase7. butyryl-acyl-carrier peptide + condensing-enzyme domain butyryl-condensing enzyme + acyl-carrier peptide thioesterase_____________________________________________________________________________________________________________________
7 enzymatic activities + 1 acyl carrier protein
Multi-domain gene assembly
Principal biochemical reactions in the synthesis of Principal biochemical reactions in the synthesis of fatty acidsfatty acids from from malonyl CoAmalonyl CoA in in eukaryotes and eubacteriaeukaryotes and eubacteria_____________________________________________________________________________________________________________________
Reaction Enzyme_____________________________________________________________________________________________________________________1. acetyl CoA + condensing-enzyme domain acetyl-condensing enzyme acetyl transferase2. malonyl CoA + acyl-carrier peptide malonyl-acyl-carrier peptide malonyl transferase3. acetyl-condensing enzyme + malonyl-acyl-carrier peptide -ketoacyl-carrier peptide -ketoacyl synthase-keto-acyl carrier peptide + NADPH + H+ -hydroxyacyl-carrier peptide + NADP+ -ketoacyl reuctase5. -hydroxyacyl-carrier peptide 2-butenoyl-acyl-carrier peptide + H2O -hydroxyacyl
dehydratase6. 2-butenoyl-acyl-carrier peptide + NADPH + H+ butyryl-acyl-carrier peptide + NADP+ enoyl reductase7. butyryl-acyl-carrier peptide + condensing-enzyme domain butyryl-condensing enzyme + acyl-carrier peptide thioesterase_____________________________________________________________________________________________________________________
In most bacteria, these functions are carried on by discrete monofunctional proteins.
Multi-domain gene assembly
Principal biochemical reactions in the synthesis of Principal biochemical reactions in the synthesis of fatty acidsfatty acids from from malonyl CoAmalonyl CoA in in eukaryotes and eubacteriaeukaryotes and eubacteria_____________________________________________________________________________________________________________________
Reaction Enzyme_____________________________________________________________________________________________________________________1. acetyl CoA + condensing-enzyme domain acetyl-condensing enzyme acetyl transferase2. malonyl CoA + acyl-carrier peptide malonyl-acyl-carrier peptide malonyl transferase3. acetyl-condensing enzyme + malonyl-acyl-carrier peptide -ketoacyl-carrier peptide -ketoacyl synthase-keto-acyl carrier peptide + NADPH + H+ -hydroxyacyl-carrier peptide + NADP+ -ketoacyl reuctase5. -hydroxyacyl-carrier peptide 2-butenoyl-acyl-carrier peptide + H2O -hydroxyacyl
dehydratase6. 2-butenoyl-acyl-carrier peptide + NADPH + H+ butyryl-acyl-carrier peptide + NADP+ enoyl reductase7. butyryl-acyl-carrier peptide + condensing-enzyme domain butyryl-condensing enzyme + acyl-carrier peptide thioesterase_____________________________________________________________________________________________________________________
In fungi, the activities are distributed between two proteins encoded by two unlinked intronless genes, FAS1 and FAS2. FAS1 encodes 3 of the 8 activities (-ketoacyl synthase, -ketoacyl reductase, and acyl-carrier protein). FAS2 encodes the rest of the five enzymatic activities.
Multi-domain gene assembly
In animals, all functions are performed by one polypeptide, fatty-acid synthase. The gene product has 8 modules, including one that performs a dual function and another whose function is unrelated to fatty-acid synthesis but may determine the 3D structure of this protein.