Protein Biosynthesis (Translation) Zhongren Ding, MD, PhD Department of Biochemistry Zhongren Ding (...

Protein Biosynthesis(Translation)

Zhongren Ding, MD, PhD

Department of Biochemistry

Zhongren Ding ( 丁忠仁 )

Tel: 5423 7896

Email: dingzr@fudan.edu.cn

Office: Rm 312, 7# Building

Lab: Rm 318, 7# Building

Protein Biosynthesis

Protein biosynthesis: easy, you can do it!

How is protein biosynthesized? Understand the process of protein

synthesis, regulation and control with aim to interfere protein synthesis.

1. Stop abnormal protein synthesis (tumor treatment).

2. Inhibit bacteria protein synthesis, treat infectious diseases;

3. Biosynthesis of protein drugs.

Not easy

Protein Chemical (Artificial) Synthesis

Chemical synthesis of crystalline bovine insulin (51 aa, Shanghai, China 1965 ）

Chemical synthesis of peptide (mg – kg, 140 aa, purity: 98 ％ , RMB100/aa)

G13 blocking peptide inhibits human platelet aggregation and ATP release

Synthesized peptide AYPGKF 100 microM stimulates human platelet aggregation and ATP release in the presence of aspirin

• Grasp the properties of genetic code;

• Familiar with the structure and function of mRNA, tRNA, and ribosome;

• Familiar with the process of protein synthesis in prokaryotes;

• Familiar with the process of protein synthesis in eukaryotes;

• Know protein folding, and clinical relevance of protein synthesis.

Purpose and Requirements

1. What is protein biosynthesis? Why also called translation?2. Protein synthesis system (What is needed to synthesize protein) 3. Protein synthesis process 4. Posttranslational processing and targeting5. Protein synthesis in medicine

1. What is protein biosynthesis? Why also called translation?

• A process in which amino acids are synthesized into polypeptide in ribosomes using mRNA as template; also includes folding, posttranslational processing, transportation.

• In protein synthesis, the genetic information present in mRNA is transformed into the sequence of amino acids of the corresponding specific protein, similar to the process one language translated into another language, so it’s also called “translation”.

– Amino acids: materials– mRNA: template, consisting of genetic codes– tRNA: transfer tool and adaptor– Ribosome: workplace– Protein factors– Synthetic enzymes

2. Protein synthesis system (What is needed to synthesize protein?)

2. Protein synthesis system

2.1 Genetic code defines the relationship between the base sequence of mRNA and the amino acid sequence of polypetide

Three bases needed to specify one amino acid, because:

1) 4 bases need to code 20 amino acids,

2) 41 = 4, 42 = 16, 43 = 64

The 3 bases triplets on mRNA (and DNA) coding for a specific amino acid is called genetic codes.

Sydney Brenner (1927 - )

2002 Nobel Prize Laureate

Genetic Codes and its prominent properties

Start codon: AUG (also codes methionine)

Stop codon: UAA, UAG, UGA

Degenerate: 2 – 6 codons specify one amino acid except for AUG and UGG (coding for tryptophan)

Reading frame of mRNA and the importance of the start and stop codon

Reading frame: a contiguous and nonoverlapping set of three-nucleotide condons in mRNA as well as DNA.

Open Reading Frame (ORF): reading frame starting with start condon and ending with stop condon.

PPP5 3

protein

Noncoding sequence Ribosome binding site

Start codon Stop codonCoding sequence

PPPmG -5 3

protein

AAA …

mRNA in eukaryotes is usually monocistronic: one mRNA encodes only a single polypeptide chain.

mRNA in prokaryotes usually encodes more than one polypeptide chain. This is called polycistronic.

Polycistron and Monocisctron

A) It is colinear and directional: mRNA is read from 5’ to 3’, protein is synthesized from N terminus to C terminus.

Properties of genetic code

Colinear: amino acid sequence N to C corresponds exactly to the sequecne of their codons in the mRNA read 5’ to 3’.

N CPolypeptide extending direction

5′ 3′Codon reading

Polypeptide chain Stop codon

PCR primer design: 5’ to 3’ for both forward and reverse primer; for protein expression, forward primer must contain start codon.

B) It is nonoverlapping and “commaless” : No overlapping (each nucleotide is used only once ) and no empty spaces in between in the coding sequence.

Insertion or deletion leading to frameshift mutation

C) It contains 61 amino acid coding codons and 3 stop condons UAA, UAG, and UGA. Methionine coding codon AUG is also used as start condon.

Application:

tag and truncating mutation (insert or mutate into a stop codon)

D) It is unambiguous. Each codon specifies one and only one amino acid.

E) It is degenerate: one amino acid has more than one codon.

One of the consequences of degeneracy is that a mutation which produces a base change in DNA may not result in an amino acid change in the encoded protein.

Synonyms: refers to the codons for the same amino acid. e.g. GUU, GUC, GUA, GUG represent for Val.

Degeneracy of genetic codes

F) It is universal: the genetic code system is used by all living organisms with the minor exception:

in cytosol in mitochondriaAUA Ile Met

UGA Stop Trp

AGA Arg Stop (animal)

CGG Arg Trp (plant)

CUN Leu Thr (yeast)

The near-universality of genetic code means:

1. all surviving life on earth is descended from a common ancestor;

2. eukaryotic proteins can be expressed in prokaryotic cells.

G) Each mRNA sequence can be read in three possible reading frames, start condon AUG at 3’-end determines the right reading frame.

Reading frame 1: UUA UGA GCG CUA AAU

Leu Stop Ala Leu Asn

Reading frame 2: U UAU GAG CGC UAA AU

Tyr Glu Arg Stop

Reading frame 3: UU AUG AGC GCU AAA U

Met Ser Ala Lys

H) Wobble base pairing: Base pairing between the 3’ position of the codon and 5’ position of the anticodon may occur by a non-standard way. This allows one tRNA to recognize more than one codon.

61 sense codons, 40 – 50 tRNAs with different anticodons

“2 out of 3 reading”

2.2 tRNA is the adptor and amino acid

carrier in protein synthesis Common structural and functional features: Structural features:

• Small RNA, about 80 nucleotides long;

• cloverleaf-like: amino acid binding site at 3’-end (binding to the ribose of the last nucleotide) and anticodon arm.

Functional features:• Adaptor: the anticodon of tRNA

guarantees AA is placed correctly on mRNA

• Carrier: AA is attached at 3’-end (-CCA)

• D arm and TψC arm participate in recognition by aminoacyl-tRNA synthetase and in binding to ribosome

Secondary structure of tRNA

Mahlon Hoagland(1921 - 2009)

tRNA discoverer

Francis Crick (1916 - 2004)

Adptor hypothesis

tRNA is the adptor and amino acid carrierTertiary structure of tRNA

James Watson (1928 - )

Nature 1953, 1 page, 1 figuretRNA adaptor hypothesis

Watson:• Ideas are more important than facts.• New ideas need new facts.• Never be in a room where you are the

brightest person.• Choose a field ahead of time but

expecting to get an answer in reasonable time

2.3 Ribosomes are the workbenches

for protein synthesis.

Consist of 2 subunits: large subunit and small subunit;

2 components: RNA (rRNA) and and protein with rRNA as major component (> 50%)

Structure of ribosome: two subunits, 3 sites* (location and role)

Largesubunit

smallsubunit

E site P site A Site

Binding site

*Eukaryotic ribosome has no E site

2.4 Protein factors are required in protein synthesis Soluble cytoplasmic proteins including:

• initiation factor

• elongation factor

• termination factor

Usually belong to G protein family using energy released upon

hydrolysis of bound GTP to fuel switch-like conformation

change.

A.To start protein synthesis, the ribosome binds to a site near the 5’ terminus of mRNA.

B.mRNA is read in the 5’ to 3’ direction, whereas the polypeptide is synthesized in the amino to carboxyl terminal direction (colinear)

3. Protein synthesis process

3.1 Features of ribosome protein synthesis:

C. Ribosome 3 sites: P site for tRNA bearing growing polypeptide and A site for incoming aminoacyl-tRNA. E site is the exit site for prokaryotic ribosome.

D. The ribosome forms the peptide with peptidyl-tRNA and aminoacyl-tRNA are bound to P site and A site.

3.1 Features of ribosome protein synthesis:

Largesubunit

smallsubunit

E site P site A Site

Binding site

*Eukaryotic ribosome has no E site

E. Energy-dependent steps in ribosome protein synthesis requires GTP rather that ATP (ATP is required in amino acid activation).

F. Each ribosome synthesize only one polypeptide at a time, but one mRNA is read simultaneously by many ribosomes.

3. Protein synthesis process3.1 Features of ribosome protein synthesis:

polysome

• Amino acid activation• Initiation• Elongation• Termination• Release

Protein biosynthesis takes place in stages:

3. Protein synthesis process3.2 Activation of amino acid (the process amino acid is attached to

the 3’-end of tRNA):

Aminoacyl-AMP

Amino acid + tRNA aminoacyl- tRNAATP AMP ＋ PPi

aminoacyl-tRNA synthetase

Two steps:

Step 1: AMP added.

Step 2: AMP removed and tRNA added to amino acid

• 20 different aminoacyl- tRNA synthetases for 20 aa;• 40+- different tRNA;• Aminoacyl-tRNA synthetase can recognize several tRNA, but is highly selective for the AA.

Mechanisms for correct attachment of amino acid to tRNA

1. Aminoacyl tRNA synthetase is highly specific for one amino acid and the corresponding tRNA;

2. Aminoacyl tRNA synthetase has proofreading function:

(1) hydrolyze aminoacyl-AMP from the enzyme;

(2) hydrolyze aminoacyl-tRNA.

3.2 Activation of amino acid

MetO tRNAf

HN CHHC

O tRNAf

HN CHHC

OSynthetase transformylase

tRNA + Met

(Met-tRNAiMet) (fMet-tRNAiMet )

N10-formyl tetrahydrofolate tetrahydrofolate

H2N CH

O tRNAfMet

H2N CH

O tRNAfMet

Two types of tRNA specific for methionine:

• bacteria: tRNAmet (for AUG condons at interior position) and

tRNAfmet (for initiation AUG condon)

• eukaryotes: tRNAmet (for AUG condons at interior position) and

tRNAimet (for initiation AUG condon)

Two successive reactions to form N-formylmethionyl-tRNA

3. Protein synthesis process3.2 Activation of amino acid

A process that mRNA 、 initiation aminoacyl-tRNA bind to ribosome and form the translation (protein synthesis) initiation complex.

3. Protein synthesis process3.3 Initiation of peptide chain synthesis

⑴ Small subunit dissociates

from large subunit upon IF1 and IF3 binding to small subunit;⑵ mRNA binds to small subunit via S-D

sequence base paring with the seq on 16S RNA;

⑶ Initiation aminoacyl- tRNA binds to small unit via anticodon-AUG paring ；

⑷ Large subunit binds to small subunit.

4 steps to form translation initiation complex:

IF-3IF-1

⑴ Dissociation of ribosome large and small subunits

A U G5' 3'

⑵ mRNA locating and binding on small subunit

Shrine-Dalgarno sequence ： a conserved short sequence (usually AGGAGGU) about 10 nucleotides upstream of the AUG start codon of prokaryotic mRNA. By base pairing with a complementary short sequence at 3’ end of 16S rRNA of ribosome small subunit (usually UCCUCC) ， ribosome is correctly located on mRNA (correct AUG is therefore selected to initiate protein sysnthesis ） .

Shine J, Dalgarno L. Nature 1975

Two mechanisms involved in locating and binding of mRNA on small subunit of eukaryotic ribosome ：(1) Shine-Dalgarno sequence (S-D sequence);(2) Small nucleotide sequence down stream of S-D sequence

S-D sequence

16S-rRNA of small subunit of ribosome

rpS-1 recognization seq

IF-2 GTP

(3) Initiation aminoacyl tRNA (fMet-tRNAfMet ) binding to small

subunit

A U G5' 3'

IF-2 GTPGDPPi

⑷ Binding of large subunit to the 30S pre-initiation complex leading to the formation of 70S initiation complex

A U G5' 3'

IF-3IF-1

A U G5' 3'

IF-2 GTPIF-2 -GTPGDP

The process of initiation complex formation

A process that polypeptides grow stepwise from amino terminus to the carboxyl terminus.

3. Protein synthesis process3.4 Elongation of peptide chain synthesis

⑴ Positioning / registration: Aminoacyl-tRNA gets into the A site of ribosome along with GTP-bound elongation factor Tu (EF-Tu-GTP)

⑵ Peptide bond formation: peptidyl transferase (23S rRNA of large subunit) catalyzes the formation of peptide bond; no energy needed at this step.

⑶ Translocation: peptidyl-tRNA translocates from A site to P site. Hydrolysis of EF-G bound GTP provides energy.

3 steps to add one amino acid:

Positioning

Tu TsGTP

Aminoacyl-tRNA gets into A site guided by the genetic codon on A site

GTPTu GDP

GTPfMet

Positioning

Peptide bond formation

A process of peptide bond formation catalyzed by peptidyl transferase (the activity of 23S rRNA of large subunit)

Translocation

With the acting of translocase EF-G, ribosome moves along mRNA by 3 bases to 3’ end, therefore, the newly formed peptidyl-tRNA and mRNA tranlocates from A site to P site, the

discharged tRNA enters into E site

Tu GTP

Translocation

positioning

Peptide bond formation

translocation

Start codon 2nd condon

Ribosome cycle

Release factors terminate the ribosome cycle.

3. Protein synthesis process3.5 Termination of peptide chain synthesis

① Ribosome cycle continues and polypeptide become longer and longer until a stop codon appears at A site of ribosome.

② No tRNA recognizes and base pairs stop codon, while release factors (RF-1, RF-2, RF-3) do it.

③ After binding of RF to stop condon, release factors induce the cleavage of the bond between polypeptide and tRNA.

④ Polypeptide, tRNA and mRNA are released, ribosome dissociates into small and large subunits.

At least 4 high-energy bonds are consumed to add one amino acid (to form a peptide bond):

– 2 high-energy bonds in ATP for the activation of amino acid (the formation of aminoacyl-tRNA);

– 2 high-energy bonds from GTP for positioning (placement) and translocation in elongation step;

– 1 high-energy bonds from GTP for the formation of initiation complex (one-time expense).

3.6 Protein synthesis is energetically expensive

Generally similar but more complex compared with that in prokaryotic cells, the most significant difference is the initiation step:

⑴ Small subunit dissociates from large subunit upon eIF1 and eIF3 binding to small subunit;

⑵⑵ eIF2-GTP-eIF2-GTP-Met-tRNAiMet-tRNAiMetMet binds to small subunit binds to small subunit to form 43S to form 43S preinitiation complex;preinitiation complex;

⑶⑶ eIF4 protein group transfers eIF4 protein group transfers mRNA to small subunitmRNA to small subunit, , which migrate and scan mRNA to find the start codonwhich migrate and scan mRNA to find the start codon ；；

⑷ With the acting of eIF5, GTP hydrolysis and release of all eIFs, large subunit binds to small subunit.

3. Protein synthesis process3.7 Protein synthesis in eukaryotic cells

MetMet

40S40S

60S60S

MetMet

40S40S

60S60S

eIF-1AeIF-1A 、、 eIF-3eIF-3 、、 ①

GDP+Pi

Release all elFRelease all elFelF-5④

ADP+Pi

elF4 group proteins③Met

Met-tRNAiMet-elF-2 -GTP

Process of translation initiation complex in eukaryotes

4. Posttranslational processing and targeting

4.1 Newly synthesized polypeptide chains undergo folding• Most newborn polypeptide are non-

functional, need to be folded to form higher ordered structure;

• Some polypeptides fold spontaneously, while most need assistance by some helper proteins called molecular chaperones which provide a protective (hydrophobic) environment for peptide folding;

• Most chaperones are heat shock protein (HSP)

• Noncovalent interactions participate in protein folding.

• Protein disulfide isomerase (PDI) patterns disulfide bonds and affect protein folding

• Peptidyl prolyl isomerase (PPI) catalyzes peptidyl prolyl isomerization as a rate-limiting step .

• One third proteins failed to fold properly; misfolded proteins are rapidly degraded.

4. Posttranslational processing and targeting4.2 Newly synthesized polypeptide chains undergo

posttranslational (covalent) modification and processing

4.2 Newly synthesized polypeptide chains undergo posttranslational (covalent) modification and processing

(1) Amino-terminal modification: removal of formyl group, Met residue, signal peptide;

(2) Modification on individual amino acids: I. Disulfide bridge between cysteins in intrachain and

interchain;

(2) Modification on individual amino acids: II. Glycosylation: O-linked glycosylation (oligosaccharide

attached to the oxygen of serine and threonine) and N-linked glycosylation (oligosaccharide attached to the nitrogen of asparagine in N-X-S/T consensus);

4.3 Most newly synthesized polypeptide chains are tranlocated to proper desstinations

(1) Three fates of newborn proteins: keep in cytosol, secrete extracellularly, translocate to specific organells (endoplasmic reticulum, mitochondria, lysosomes, nucleus).

(2) Signal peptide (signal sequence), a highly variable sequence of about 20 – 25 amino acid residues usually found at the amino end is needed for protein targeting.

(3) Usually signal peptides are cleaved off by signal peptidase after protein targeting (not for nuclear protein signal sequenc, Nuclear Localization Sequence, which can be in internal location)

Signal peptide leads protein into endoplasmic reticulum

5. Protein synthesis in medicine

5.1 Molecular diseases are associated with defective proteins

• Molecular diseases: diseases caused by genetic information alteration (sickle cell anemia, P2Y12 receptor deficiency)

• Prenatal diagnosis and treatment of molecular diseases

Hemoglobin beta chain position 6 glutamate replaced valine due to one base mutation (GAG to GUG

5.2 Protein synthesis can be inhibited by antibiotics and toxins

• Cells stop growing when protein synthesis is inhibited• Some antibiotics inhibit bacteria growth or kill them by

binding to ribosome sites specific for prokaryotic cells (bacteria), interfering bacteria (not human) protein synthesis, therefore it can be used to treat infectious diseases

puromycin Aminoacyl-tRNA

Similar structure between puromycin and aminoacyl tRNA, which binds to A site and block polypeptide elongation in both eukaryotes and prokaryotes

5.2 Protein synthesis can be inhibited by antibiotics and toxin

• Combination of antibiotics based on different targeting sites.

• Drug resistance mutations are not induced by antibiotics, antibiotics still need to be used very carefully.

• Diphtheria toxin and ricin inhibit human protein synthesis, are toxic to human.

Diphtheria toxin inactivates eEF-2 by ADP-ribosylation

Elongation factor-2

（ active ）

ADP Elongation factor-2

（ inactive ）

eEF-2 as translocase

Diphtheria toxin

1. Protein biosynthesis: N terminus to C-terminus2. DNA, RNA synthesis: 5’ to 3’3. Genetic codes read : 5’ to 3’4. Initiation codon: AUG in mRNA; ATG in DNA5. Stop condon: UAA, UAG, UGA in mRNA; TAA, TAG, TGA in DNA6. tRNA 3’-end amino acid acceptor: 5’-CCA-3’7. Signal peptide.8. Properties of gentetic codes.9. Compare the common and different points in protein synthesis process

between prokaryotes and eukaryotes.

Homework• Read Principal Points on page 232 and Summary on page 252 – 22;

• Answere the following questions:

Thank you!

Release Factor is a molecular mimic of a tRNA

eRF1 tRNA

Hollopeter et al, Nature 2001

Nurden et al, J Clin Invest 1995

Excessive bleeding, after a minor accident, developed subretinal hemorrhage leading

to the loss of an eye.

Two bases deletion leading to frameshift mutation causes

platelet P2Y12 receptor deficiency and bleeding

Three important discoveries in protein biosynthesis study

Paul Zamecnik (1912- 2009)

Ribosome is the workbenchfor protein biosynthesis

Mahlon Hoagland(1921 - 2009)

Discovery of tRNA

tRNA as an adaptor

James Watson (1928 - )

Nature 1953, 1 page, 1 figure(tRNA as adaptor)

Watson:• Ideas are more important than facts.• New ideas need new facts.• Never be in a room where you are the brightest person.• Choose a field ahead of time but expecting to get an answer in

reasonable time

RSEANLQSKSED

M T L N I L P E F K Q N G D T S L

Ding et al, Am J Physiol 2005

Hollopeter et al, Nature 2001

Nurden et al, J Clin Invest 1995

Excessive bleeding, after a minor accident, developed subretinal hemorrhage leading to the

loss of an eye.

How can we take advantage of degeneracy of genetic codon?

• Insert a restriction enzyme site (site-directed mutagenesis) without changing amino acid sequence.

• Inactivate restriction enzyme site without changing amino acid sequence to facilitate enzyme digestion.

Protein Biosynthesis (Translation) Zhongren Ding, MD, PhD Department of Biochemistry Zhongren Ding (...

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