Lecture 4

Protein Biosynthesis (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 f

actor (IF), elongation factor (EF), releasing factor (RF)

• ATP, GTP, Mg2+

• Messenger RNA is the template for the pr

otein 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

5-PPP 3

protein

Non-coding ribosomal protein binding site

Starting code Stop codonCoding region

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.

• One codon for start signal: AUG. It also codes for methionine.

• Three codons for stop signal: UAA, UAG, UGA.

• 61 codons for 20 amino acids.

Genetic codon

Established the chemicalstructure oftRNA

Established the in vitro system for revealing the genetic codes

Devised methods to synthesize RNAs with definedsequences

1. Direction

• The direction of template mRNA: 5

´→ 3´end

Properties of genetic codon

•The genetic codons should be read continuously without spacing or overlapping.

2. Commaless

Open reading frame(ORF)

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

Frameshift

• Except Met and Trp, the rest amino acids have 2, 3, 4, 5, and 6 triplet codons. (synonym)

• These degenerated codons differ only on the third nucleotide.

3. Degeneracy

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

5. Wobble base pair•Although there are 61 codons for amino acids, the number of tRNA is far less (around 40).

•A single tRNA can recognize more than one codon.

•Non-Watson-Crick base pairing is permissible between the 3rd nucleotide of the codon on mRNA and the 1st nucleotide of the anti-codon on tRNA.

　

Base-pair of codon and anticodon

tRNA

§1.2 tRNA and AA Activation

　　　　

Activation of amino acid

Ala-tRNAAla

Ser-tRNASer

Met-tRNAMet

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 proo

freading 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

For prokaryotes: • fMet-tRNAi

met can only be recognized by initiation codon.

• Met-tRNAemet is used for elongation.

For eukaryotes: • Met-tRNAi

met is used for initiation. • Met-tRNAe

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.

Three Three rRNA5252 proteins

Four Four rRNA8383 proteins

• Ribosomes are ribonucleoprotein particles for synthesizing proteins.

The Nobel Prize in Chemistry 2009"for studies of the structure and function of the ribosome"

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

Section 2

Protein Synthetic Process

General concepts

• The direction of template mRNA: 5´→ 3´end

• The direction of the protein synthesized : N-terminal→C-terminal

• 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-tRNAi

met on the P site – Associating the 50S subunit

• Three initiation factors: IF-1, IF-2 and IF-3.

Prokaryotic initiation

IF-3IF-1

• The IF-1 and IF-3 bind to the 30S subunit, making separation between 50S and 30S subunit.

Initiation 1: Separation between 50S and 30S subunit

A U G5' 3'

IF-3

IF-1

Initiation 2: Positioning mRNA on the 30S subunit

• The mRNA then binds to 30S subunit.

Shine-Dalgarno (S-D) sequence in mRNA

-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, RBS). •S-D sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon.

IF-3

IF-1

IF-2 GTP

A U G5' 3'

Initiation 3: Registering fMet-tRNAimet on the P site

• The complex of the GTP-bound IF-2 and the fMet-tRNA enters the P site.

IF-3

IF-1

IF-2 GTPGDPPi

A U G5' 3'

Initiation 4: Associating the 50S subunit

• 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 。

§2.2 Elongation

• Elongation involves the addition of amino acids to the carboxyl end of the growing polypeptide chain.

• Three steps in each cycle:– Entrance: positioning an aminoacyl-tRNA in

the A site– Peptide bond formation: forming a peptide b

ond– Translocation: translocating the ribosome to

the next codon• 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.

Step 3: Translocation• EF-G is a translocase. • GTP bound EF-G provides the energy to move the ribo

some one codon toward the 3’ end on mRNA.• After the translocation, the uncharged tRNA is releas

ed from the E site.

fMet

A U G5' 3'

fMet

Tu GTP

Peptide bond formation

Elongation

Translocation

Entrance

§2.3 Termination

• Prokaryotes have 3 release factors: RF-1, RF-2 and RF-3. – RF-1 and RF-2: Recognize the terminat

ion codons• RF-1: Recognizes UAA, UAG• RF-2: Recognizes UAA, UGA

– RF-3: GTP hydrolysis and stimulates the activity of RF-1 and RF-2

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

AA activation ： two ~P bonds

initiation ： one GTP (IF-2-GTP)

elongation ： two GTP (Tu-GTP, EF-G-GTP)

termination ： one GTP (RF-3)

Total: at least four high-energy bonds per peptide bond are consumed.

Translation of prokaryotes

• 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

eIF2 Facilitates 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 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

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 eIF4

F complex

eIF5Promotes dissociation of several other IFs from 40S subunit as a pr

elude 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

Eukaryotic elongation

• Elongation factors are EF-1 (EF-T) and EF-2 (EF-G).

• There is no E site on the ribosome.

Termination

• Eukaryotes have only 1 releasing factor: eRF.

•Proteins are synthesized on a single strand mRNA simultaneously, allowing highly efficient use of mRNA.

Polysome

Section 3

Protein Modification and Protein

Targeting

• Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino acids.

• 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.

chaperonins GroEL and GroEs pathway in E. coli

non-folded peptide folded peptide

GroEL

GroES

Chaperonin•The structure of these chaperonins resemble two donuts stacked on top of one another to create a barrel.

§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

§3.3 Modification of spatial structure

• Assemble of subunits: Hb• Attachment of prosthetic groups: glycopr

oteins• Connection of hydrophobic aliphatic chai

ns

• 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-

Secretory protein into ER

SRP: signal recognition particle

The Nobel Prize in Physiology or Medicine 1999

• for the discovery that "proteins have intrinsic signals that govern their transport and localization in the cell."

Günter Blobel

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 interference interact specifically with proteins and RNAs to interrupt the protein synthesis.

5' 3'

P Asite site

chloromycetin

streptomycin and karamycin

Puromycin

Tetracycline

cycloheximide

Antibiotics

(block the A site to prevent binding of AA-tRNA with 30S)

(similar to Tyr-tRNA, release the prematured peptide)

(repress the translocase, inhibit the

elongation, for eukaryotic organisms, 60S )

(block the peptidyl transferase, and inhibit the elongation, 50S)

(bind to 30S subunit, interfering with the binding

of fMet-tRNA to the 30S, or misread mRNA )

name target functiontetracycline 30S block the A site to prevent

binding of AA-tRNA with 30S

streptomycin 30S bind to 30S subunit, interfering with the binding of fMet-tRNA to the 30S, or misread mRNA

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

Puromycin

• It has a similar structure to Tyr-tRNA.

• release the prematured peptide.

• It works for both prokaryotes and eukaryotes.

Points Ⅰ. Genetic code• Triplet, comma free, non-overlapping, universal, deg

enerate, start and stop signals, wobble in the tRNA anti-codon

Ⅱ. Components required for translation 1. Amino acids 2. tRNA 3. Aminoacyl-tRNA synthetase 4. mRNA: template for the protein synthesis 5. Small and large ribosomal subunits: A, P, and E site

s 6. Protein factors, like IF, EF, and RF 7. ATP, GTP

Ⅲ. Steps in Prokaryotic protein synthesis 1. Charging of tRNA aminoacyl-tRNA synthetase2. Initiation SD sequence, IF-1, IF-2, IF-3, 30s and 50s ribosomal su

bunit, fMet-tRNAfMet , 70s initiation complex, GTP3. Elongation a. fMet-tRNA in P site. New amino-acyl tRNA to A site,

EF-Tu and EF-Ts. GTP. b. Peptidyltranferase: forms a peptide bond c. Translocation 4. Termination Stop codons, RF-1, RF-2, RF-3 .5. PolysomeⅣ. Post-translational modification:

– Protein folding, Protein targeting Ⅴ. Interference of Translation: Antibiotics