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, Mg2+

• 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-coding ribosomal protein binding site

Starting code Stop codonCoding region

5-PPP 3

protein

PPP5-mG - 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.

Genetic codon

• 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 U A A5 ' 3 '

ORF

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

Frameshift

2. Degeneracy

• 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. 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.

　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: te

rmination

Mitochondria

• AUA: Met, initiation• UGA: Trp• AGA, AGG: termina

tion

tRNA

§1.2 tRNA and AA Activation

Activation of amino acid

　

　　　

Ala-tRNAAla

Ser-tRNASer

Met-tRNAMet

Activated amino acid

• Active formaminoacyl － tRNA

• Activation site- carboxyl group

• Linkageester bond

• Activation energy2 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-tRNAmet can be formylated to fMet-tRNAi

met.

Prokaryotic Met-tRNAmet

Met-tRNAmet + N10-formyl tetrahydrofolate

fMet-tRNAimet + tetrahydrofolate

formyl transferase

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

§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

70S ribosome

50S large subunit

23S rRNA 5S rRNA

31 proteins

16S rRNA

21 proteins30S small subunit

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

location function

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 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

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-3IF-1

A U G5' 3'

IF-2 GTPIF-2 -GTPGDP

Pi

One GTP is consumed in initiation course 。

• 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

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

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

§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.

Tu TsGTP

GDP

A U G5' 3'

Tu

Ts

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.

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.

Translocation

fMet

A U G5' 3'

fMet

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

U A G5' 3'

RF

COO-

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.

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.

Mechanism

§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

target signal

Nucleus Nuclear Location Sequence

Peroxisome ----SKL-COO-

Mitochondria 20-35 AA at N-terminus

Endoplasmic reticulum

----KDEL-COO-

a. Secretory protein

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

N-terminus hydrophobiccore

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 20-25 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.

5' 3'

P Asite site

chloromycetin

streptomycin and karamycin

Puromycin

Tetracycline

cycloheximide

Antibiotics

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

• 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

Diphtheria toxin

EF-2

active

+

Diphtheria toxin

O

OH OH

CH2OADP N

CNH2

O

O

OH OH

CH2OADP

EF-2

(inactive)

N

CNH2

O

+

• Interferons are cytokines produced during immune response to antigens, especially to viral infections.

Interferon

InterferondsRNA

protein kinase

eIF2 eIF2-P

ATP ADP

active inactive

Interferon

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