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Transcript of Chapter 12 Translation. The synthesis of protein molecules using mRNA as the template, in other...
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Chapter 12
Translation
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The synthesis of protein molecules using mRNA as the template, in other word, to translate the nucleotide sequence of mRNA into the amino acid sequence of protein according to the genetic codon.
Translation
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Section 1
Protein Synthetic System
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Protein synthesis requires multiple elements to participate and coordinate.
• mRNA, rRNA, tRNA
• substrates: 20 amino acids
• Enzymes and protein factors: initiation factor (IF), elongation factor (EF), releasing factor (RF)
• ATP, GTP, Mg2+
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• Messenger RNA is the template for the protein synthesis.
• Prokaryotic mRNA is polycistron, that is, a single mRNA molecule may code for more than one peptides.
• Eukaryotic mRNA is monocistron, that is, each mRNA codes for only one peptide.
§1.1 Template and Codon
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polycistron
monocistron
Non-coding ribosomal protein binding site
Starting code Stop codonCoding region
5-PPP 3
protein
PPP5-mG - 3
protein
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Genetic codon
• Three adjacent nucleotides in the 5´-3´ direction on mRNA constitute a genetic codon, or triplet codon.
• One genetic codon codes for one amino acid.
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Genetic codon
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• Three codons for stop signal: UAA, UAG, UGA.
• One codon for start signal: AUG. It also codes for methionine.
• 61 codons for 20 amino acids.
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Properties of genetic codon
1. Commaless
A complete sequence of mRNA, from the initiation codon to the termination codon, is termed as the open reading frame.
A U G U A A5 ' 3 '
ORF
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The genetic codons should be read continuously without spacing or overlapping.
U U C U C G G A C C U G G A G A U U C A C A G U
commaless
U U C U C G G A C C U G G A G A U U C A C A G U
spacing
overlapping
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Frameshift
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2. Degeneracy
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• Except Met and Trp, the rest amino acids have 2, 3, 4, 5, and 6 triplet codons.
• These degenerated codons differ only on the third nucleotide.
GCU ACU GCC ACC GCA ACA GCG ACG
Ala Thr
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3. WobbleNon-Watson-Crick base pairing is permissible between the third nucleotide of the codon on mRNA and the first nucleotide of the anti-codon on tRNA.
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Base-pair of codon and anticodon
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4. Universal
The genetic codons for amino acids are always the same with a few exceptions of mitochondrial mRNA.
Cytoplasm
• AUA: Ile• AUG: Met, initiation• UAA, UAG, UGA: te
rmination
Mitochondria
• AUA: Met, initiation• UGA: Trp• AGA, AGG: termina
tion
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tRNA
§1.2 tRNA and AA Activation
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Activation of amino acid
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Ala-tRNAAla
Ser-tRNASer
Met-tRNAMet
Activated amino acid
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• Active formaminoacyl - tRNA
• Activation site- carboxyl group
• Linkageester bond
• Activation energy2 high-energy bonds
Summary of AA activation
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• Aminoacyl-tRNA synthetase has the proofreading ability to ensure that the correct connection between the AA and its tRNA.
• It recognizes the incorrect AA, cleaves the ester bond, and links the correct one to tRNA.
Protein synthesis fidelity
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• Prokaryotic Met-tRNAmet can be formylated to fMet-tRNAi
met.
Prokaryotic Met-tRNAmet
Met-tRNAmet + N10-formyl tetrahydrofolate
fMet-tRNAimet + tetrahydrofolate
formyl transferase
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For prokaryotes:
• fMet-tRNAimet can only be recognized
by initiation codon.
• Met-tRNAemet is used for elongation.
For eukaryotes: • Met-tRNAi
met is used for initiation.
• Met-tRNAemet is used for elongation.
Initiation tRNA
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§1.3 Ribosomes
• Ribosome is the place where protein synthesis takes place.
• A ribosome is composed of a large subunit and a small subunit, each of which is made of ribosomal RNAs and ribosomal proteins.
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Molecular components of ribosome of prokaryotes
70S ribosome
50S large subunit
23S rRNA 5S rRNA
31 proteins
16S rRNA
21 proteins30S small subunit
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Ribosome of prokaryotes
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Aminoacyl site
(A site)
Composed by large and small subunit
Accepting an aminoacyl-tRNA
Peptidyl site
(P site)
Composed by large and small subunit
Forming the peptidyl bonds
Exit site (E site)
Only on large subunit
Releasing the deacylated tRNA
location function
Three sites on ribosomes
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A site, P site and E site
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Section 2
Protein Synthetic Process
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General concepts
• The direction of the protein synthesized : N-terminal→C-terminal
• The direction of template mRNA: 5´→ 3´end
• The process of Protein :
initiation
elongation
termination
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§2.1 Initiation
• Four steps: – Separation between 50S and 30S subuni
t– Positioning mRNA on the 30S subunit
– Registering fMet-tRNAimet on the P site
– Associating the 50S subunit
• Three initiation factors: IF-1, IF-2 and IF-3.
Prokaryotic initiation
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Shine-Dalgarno (S-D) sequence
-AGGA PuPuUUUPuPu AUG-
• purine rich of 4-9 nts long• 8-13 nts prior to AUG
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Alignment of 16S rRNA
The 3´end of 16s rRNA has consensus sequence UCCU which is complementary to AGGA in S-D sequence (also called ribosomal binding site).
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• The IF-1 and IF-3 bind to the 30S subunit, making separation between 50S and 30S subunit.
• The mRNA then binds to 30S subunit.
Initiation 1-2
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• The complex of the GTP-bound IF-2 and the fMet-tRNA enters the P site.
Initiation 3
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Initiation 4
• The 50S subunit combines with this complex.
• GTP is hydrolyzed to GDP and Pi.
• All three IFs depart from this complex.
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IF-3IF-1
A U G5' 3'
IF-2 GTPIF-2 -GTPGDP
Pi
One GTP is consumed in initiation course 。
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• Four steps: – Separation between 60S and 40S subuni
t
– binding Met-tRNAimet on the 40S subunit
– Positioning mRNA on the 40S subunit– Associating the 60S subunit
eukaryotic initiation
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Eukaryotic initiation factorsFactor Function
eIF2Facilitates binding of initiating Met-tRNAMet to 40S ribosomal
subunit
eIF2B, eIF3 First factors to bind 40S subunit; facilitate subsequent steps
eIF4ARNA helicase activity removes secondary structure in the m
RNA to permit binding to 40S subunit; part of the eIF4F complex
eIF4BBinds to mRNA; facilitates scanning of mRNA to locate the
first AUG
eIF4E Binds to the 5’ cap of mRNA; part of the eIF4F complex
eIF4GBinds to eIF4E and to poly(A) binding protein (PAB); part of
the eIF4F complex
eIF5Promotes dissociation of several other IFs from 40S subunit
as a prelude to association of 60S subunit to form 80S initiation complex
eIF6Facilitates dissociation of inactive 80S ribosome into 40S
and 60S subunits
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MetMet
40S40S
MetMet
MetMet
40S40S
60S60S
mRNA
eIF-2BeIF-2B 、、 eIF-3eIF-3 、、 eIF-6
①
elF-3elF-3
② ATP
ADP+Pi
elF4E, elF4G, elF4A, elF4B,PAB
③
Process of eukaryotic initiation
Met-tRNAiMet-elF-2 -GTP
MetMet
60S60S
GDP+Pi
elFselFselF-5④
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§2.2 Elongation
• Three steps in each cycle:– Positioning an aminoacyl-tRNA in the A
site--- Entrance– Forming a peptide bond---Peptide bond
formation – Translocating the ribosome to the next
codon---Translocation
• Elongation factors (EF) are required.
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Step 1: Entrance
• An AA-tRNA occupies the empty A site.
• Registration of the AA-tRNA consume one GTP.
• The entrance of AA-tRNA needs to activate EF-T.
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Tu TsGTP
GDP
A U G5' 3'
Tu
Ts
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Step 2: Peptide bond formation
• The peptide bond formation occurs at the A site.
• The formylmethionyl group is transferred to α–NH2 of the AA-tRNA at the A site by a peptidyl transferase.
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Peptide bond formation 1
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Peptide bond formation 2
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Step 3: Translocation
• EF-G is a translocase.
• GTP bound EF-G provides the energy to move the ribosome one codon toward the 3’ end on mRNA.
• After the translocation, the uncharged tRNA is released from the E site.
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Translocation
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fMet
A U G5' 3'
fMet
Tu GTP
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Eukaryotic elongation
• Elongation factors are EF-1 (EF-T) and EF-2 (EF-G).
• There is no E site on the ribosome.
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§2.3 Termination
• Prokaryotes have 3 release factors: RF-1, RF-2 and RF-3.
– RF-1 and RF-2: Recognizing the termination codons
– RF-3: GTP hydrolysis and coordinating RF-1/RF-2 and rpS
• Eukaryotes have only 1 releasing factor: eRF.
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Termination 1
• The peptidyl transferase is converted to an esterase.
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• The uncharged tRNA, mRNA, and RFs dissociate from the ribosome.
Termination 2
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Energy consumption
initiation : one GTP (IF-2-GTP)
AA activation : two ~P bonds elongation : two GTP (Tu-GTP, EF-GGTP)
termination :one GTP (RF-3)
Total: at least four high-energy bonds per peptide bond are consumed.
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Translation of prokaryotes
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Proteins are synthesized on a single strand mRNA simultaneously, allowing highly efficient use of mRNA.
Polysome
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Section 3
Protein Modification and
Protein Targeting
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• The macromolecules assisting the formation of protein secondary structure include– molecular chaperon– protein disulfide isomerase (PDI)– peptide prolyl cis-trans isomerase (PPI)
§3.1 Protein Folding
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Chaperons
• A group of conserved proteins that can recognize the non-native conformation of peptides and promote the correct folding of individual domains and whole peptides.
• Heat shock protein (HSP) HSP70, HSP40 and GreE family
• Chaperonin GroEL and GroES family
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Mechanism
• Protect the unfolded segments of peptides first, then release the segments and promote the correct folding.
• Provide a micro-environment to promote the correct native conformation of those peptides that cannot have proper spontaneous folding.
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Mechanism
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§3.2 Modification of primary structure
• Removal of the the first N-terminal methionine residue
• Covalent modification of some amino acids (phosphorylation, methylation, acetylation, …)
• Activation of peptides through hydrolysis
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§3.2 Modification of spatial structure
• Assemble of subunits: Hb
• Attachment of prosthetic groups: glycoproteins
• Connection of hydrophobic aliphatic chains
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• The correctly folded proteins need to be transported to special cellular compartments to exert desired biological functions.
• AAs sequence on the N-terminus that directs proteins to be transported to proper cellular target sites is called signal sequence.
§3.4 Protein Targeting
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Signal sequences
target signal
Nucleus Nuclear Location Sequence
Peroxisome ----SKL-COO-
Mitochondria 20-35 AA at N-terminus
Endoplasmic reticulum
----KDEL-COO-
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a. Secretory protein
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Signal peptide
• All the secretory proteins have the signal peptide.
• Consist of 13-36 AA in three regions • Positively charged AA at N-terminus• Hydrophobic core of 10-15 AA in the me
dial region• Small polar AA at C-terminus
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N-terminus hydrophobiccore
Signal sequence for ER
Cleavage site
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Secretory protein into ER
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b. Mitochondrial protein
• Mitochondrial proteins in cytosol are present in precursor forms.
• Signal sequence of 20-25 AA at N-terminus are rich in Ser, Thr, and basic AA.
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b. Mitochondrial protein
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c. Nuclear protein
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Section 4
Interference of Translation
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• The protein synthesis is highly regulated.
• This process can also be the primary target for many toxins, antibiotics and interferons.
• These interferants interact specifically with proteins and RNAs to interrupt the protein synthesis.
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5' 3'
P Asite site
chloromycetin
streptomycin and karamycin
Puromycin
Tetracycline
cycloheximide
Antibiotics
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name target functiontetracycline 30S block the A site to prevent
binding of AA-tRNA with 30S
streptomycin 30S repress the translocase
chloromycetin 50S block the peptidyl transferase, and inhibit the elongation
cycloheximide 60S repress the translocase, inhibit the elongation
puromycin ribosome of P and E
release the prematured peptide
Erythromycin 50S Inhibit the translocase
Antibiotics
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• It has a similar structure to Tyr-tRNA.
• It works for both prokaryotes and eukaryotes.
Puromycin
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• Some toxins, such as plant protein Ricin, is among the most toxic substance known, which acts on 60s subunits.
Toxins
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Diphtheria toxin
EF-2
active
+
Diphtheria toxin
O
OH OH
CH2OADP N
CNH2
O
O
OH OH
CH2OADP
EF-2
(inactive)
N
CNH2
O
+
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• Interferons are cytokines produced during immune response to antigens, especially to viral infections.
Interferon
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InterferondsRNA
protein kinase
eIF2 eIF2-P
ATP ADP
active inactive
Interferon
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inteferon
dsRNA
2',5'-A synthetaseA
ppp
ATP
A
ppp p p2'
5' 5'
2'
5'
A A
2',5'-A
RNaseL RNaseLinactive active
degrade
mRNA