Presentation on theme: "Chapter 12 Translation. The synthesis of protein molecules using mRNA as the template, in other word, to translate the nucleotide sequence of mRNA into."— Presentation transcript:
Chapter 12 Translation
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
Section 1 Protein Synthetic System
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, Mg 2+
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
polycistron monocistron Non-codingribosomal protein binding site Starting code Stop codon Coding region 5-PPP 3 protein PPP 5- m G - 3 protein
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.
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.
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 UAA 5 '3 '
The genetic codons should be read continuously without spacing or overlapping. commaless spacing overlapping
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
3. Wobble Non-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.
Base-pair of codon and anticodon
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: termination Mitochondria AUA: Met, initiation UGA: Trp AGA, AGG: termination
tRNA § 1.2 tRNA and AA Activation
Activation of amino acid
Ala-tRNA Ala Ser-tRNA Ser Met-tRNA Met Activated amino acid
Active form aminoacyl － tRNA Activation site - carboxyl group Linkage ester bond Activation energy 2 high-energy bonds Summary of AA activation
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
Prokaryotic Met-tRNA met can be formylated to fMet-tRNA i met. Prokaryotic Met-tRNA met Met-tRNA met + N 10 -formyl tetrahydrofolate fMet-tRNA i met + tetrahydrofolate formyl transferase
For prokaryotes: fMet-tRNA i met can only be recognized by initiation codon. Met-tRNA e met is used for elongation. For eukaryotes: Met-tRNA i met is used for initiation. Met-tRNA e met is used for elongation. Initiation tRNA
§ 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.
Molecular components of ribosome of prokaryotes
Ribosome of prokaryotes
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 locationfunction Three sites on ribosomes
A site, P site and E site
Section 2 Protein Synthetic Process
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
§ 2.1 Initiation Four steps: –Separation between 50S and 30S subunit –Positioning mRNA on the 30S subunit –Registering fMet-tRNA i met on the P site –Associating the 50S subunit Three initiation factors: IF-1, IF-2 and IF-3. Prokaryotic initiation
Shine-Dalgarno (S-D) sequence -AGGA PuPuUUUPuPu AUG- purine rich of 4-9 nts long 8-13 nts prior to AUG
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).
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
The complex of the GTP-bound IF-2 and the fMet-tRNA enters the P site. Initiation 3
Initiation 4 The 50S subunit combines with this complex. GTP is hydrolyzed to GDP and Pi. All three IFs depart from this complex.
IF-3 IF-1 AUG 5'5'3'3' IF-2 GTP IF-2 -GTP GDP Pi One GTP is consumed in initiation course 。
Four steps: –Separation between 60S and 40S subunit –binding Met-tRNA i met on the 40S subunit –Positioning mRNA on the 40S subunit –Associating the 60S subunit eukaryotic initiation
Eukaryotic initiation factors FactorFunction eIF2 Facilitates binding of initiating Met-tRNA Met to 40S ribosomal subunit eIF2B, eIF3First factors to bind 40S subunit; facilitate subsequent steps eIF4A RNA helicase activity removes secondary structure in the mRNA to permit binding to 40S subunit; part of the eIF4F complex eIF4B Binds to mRNA; facilitates scanning of mRNA to locate the first AUG eIF4EBinds to the 5’ cap of mRNA; part of the eIF4F complex eIF4G Binds to eIF4E and to poly(A) binding protein (PAB); part of the eIF4F complex eIF5 Promotes dissociation of several other IFs from 40S subunit as a prelude to association of 60S subunit to form 80S initiation complex eIF6 Facilitates dissociation of inactive 80S ribosome into 40S and 60S subunits
Met 40S Met Met 40S 60S mRNA eIF-2B 、 eIF-3 、 eIF-2B 、 eIF-3 、 eIF-6 ① elF-3 ② ATP ADP+Pi elF4E, elF4G, elF4A, elF4B,PAB ③ Process of eukaryotic initiation Met-tRNA i Met -elF-2 -GTP Met 60S GDP+Pi elFs elF-5 ④
§ 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.
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.
TuTs GTP GDP AUG 5'5'3'3' Tu Ts
Step 2: Peptide bond formation The peptide bond formation occurs at the A site. The formylmethionyl group is transferred to α–NH 2 of the AA-tRNA at the A site by a peptidyl transferase.
Peptide bond formation 1
Peptide bond formation 2
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.
fMet AUG 5'5'3'3' Tu GTP
Eukaryotic elongation Elongation factors are EF-1 (EF-T) and EF-2 (EF-G). There is no E site on the ribosome.
§ 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.
Termination 1 The peptidyl transferase is converted to an esterase.
The uncharged tRNA, mRNA, and RFs dissociate from the ribosome. Termination 2
UAG 5'5'3'3' RF COO -
Energy consumption initiation ： oneGTP (IF-2-GTP) AA activation ： two ~P bonds elongation ： two GTP (Tu-GTP, EF-G GTP) termination ： oneGTP (RF-3) Total: at least four high-energy bonds per peptide bond are consumed.
Translation of prokaryotes
Proteins are synthesized on a single strand mRNA simultaneously, allowing highly efficient use of mRNA. Polysome
Section 3 Protein Modification and Protein Targeting
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
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
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.
§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
§3.2 Modification of spatial structure Assemble of subunits: Hb Attachment of prosthetic groups: glycoproteins Connection of hydrophobic aliphatic chains
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
Signal sequences targetsignal Nucleus Nuclear Location Sequence Peroxisome ----SKL-COO - Mitochondria AA at N-terminus Endoplasmic reticulum ----KDEL-COO -
a. Secretory protein
Signal peptide All the secretory proteins have the signal peptide. Consist of AA in three regions Positively charged AA at N-terminus Hydrophobic core of AA in the medial region Small polar AA at C-terminus
Signal sequence for ER Cleavage site
Secretory protein into ER
b. Mitochondrial protein Mitochondrial proteins in cytosol are present in precursor forms. Signal sequence of AA at N- terminus are rich in Ser, Thr, and basic AA.
b. Mitochondrial protein
c. Nuclear protein
Section 4 Interference of Translation
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.
nametargetfunction tetracycline30Sblock the A site to prevent binding of AA-tRNA with 30S streptomycin30Srepress the translocase chloromycetin50Sblock the peptidyl transferase, and inhibit the elongation cycloheximide60Srepress the translocase, inhibit the elongation puromycinribosome of P and E release the prematured peptide Erythromycin50SInhibit the translocase Antibiotics
It has a similar structure to Tyr- tRNA. It works for both prokaryotes and eukaryotes. Puromycin
Some toxins, such as plant protein Ricin, is among the most toxic substance known, which acts on 60s subunits. Toxins
Interferons are cytokines produced during immune response to antigens, especially to viral infections. Interferon