PROTEIN SYNTHESIS

Disampaikan
Oleh
Mohammad Hanafi, MBBS (Syd)., dr., MS.
 Protein Synthesis
• The production (synthesis) of proteins.
• 3 phases:
1. Transcription
2. RNA processing
3. Translation
• Remember: DNA  RNA  Protein
 DNA  RNA  Protein
Nuclear
DNA membrane

Transcription
Pre-mRNA

Eukaryotic RNA Processing

Cell mRNA

Ribosome

Translation

Protein
DNA  RNA  Protein
DNA

Transcription

mRNA

Ribosome

Translation

Protein

Prokaryotic Cell
 Question:
• How does RNA (ribonucleic acid) differ
from DNA (deoxyribonucleic acid)?
 RNA differs from DNA
1. RNA has a sugar ribose
DNA has a sugar deoxyribose

2. RNA contains uracil (U)

DNA has thymine (T)

3. RNA molecule is single-stranded

DNA is double-stranded
 1. Transcription
Nuclear
DNA membrane

Transcription
Pre-mRNA

Eukaryotic RNA Processing

Cell mRNA

Ribosome

Translation

Protein
 1. Transcription
• The transfer of information in the nucleus from a
DNA molecule to an RNA molecule.
• Only 1 DNA strand serves as the template
• Starts at promoter DNA (TATA box)
• Ends at terminator DNA (stop)
• When complete, pre-RNA molecule is released.
 Question:
• What is the enzyme responsible
for the production of the RNA
molecule?
 Answer: RNA Polymerase
• Separates the DNA molecule by breaking the H-
bonds between the bases.
• Then moves along one of the DNA strands and
links RNA nucleotides together.
 1. Transcription
DNA

RNA Polymerase

pre-mRNA
2) free mRNA nucleotides match up to the
exposed nucleotides on the DNA strand
 Question:
• What would be the complementary
RNA strand for the following DNA
sequence?

• DNA 5’-GCGTATG-3’
Complementary Base Pairs

14
 Answer:

• DNA 5’-GCGTATG-3’ 1
• RNA 3’-CGCAUAC-5’ 2

1. Template
2. mRNA
 2. RNA Processing
Nuclear
DNA membrane

Transcription
Pre-mRNA

Eukaryotic RNA Processing

Cell mRNA

Ribosome

Translation

Protein
 2. RNA Processing
• Maturation of pre-RNA molecules.
• Also occurs in the nucleus.
• Introns spliced out by splicesome-enzyme
and exons come together.
• End product is a mature RNA molecule that
leaves the nucleus to the cytoplasm.
 2. RNA Processing
pre-RNA molecule
exon intron exon intron exon

intron intron

exon exon exon

splicesome splicesome

exo exon exon

n
Mature RNA molecule
 Types of RNA
• Three types of RNA:
A. messenger RNA (mRNA)
B. transfer RNA (tRNA)
C. ribosome RNA (rRNA)

• Remember: all produced in the nucleus!

 A. Messenger RNA (mRNA)
• Carries the information for a specific protein.
• Made up of 500 to 1000 nucleotides long.
• Made up of codons (sequence of three bases:
AUG - methionine).
• Each codon, is specific for an amino acid.

4 nucleotides in mRNA so theoretically

there are 43 (64) possible combinations
of codons
Only 20 a.a.’s to encode
 A. Messenger RNA (mRNA)
start
codon

mRNA A U G G G C U C C A U C G G C G C A U A A

codon 1 codon 2 codon 3 codon 4 codon 5 codon 6 codon 7

protein methionine glycine serine isoleucine glycine alanine stop

codon

Primary structure of a protein

aa1 aa2 aa3 aa4 aa5 aa6

peptide bonds
 Genetic Codes

Codons are written 5’ to 3’

Codons for the same a.a. tend to have the nucleotides in 1st and 2nd positon
Raises the possibility of 3 different reading frames depending on the start
AUG is unique - encodes methionine
- acts as an initiation codon
 B. Transfer RNA (tRNA)
• Made up of 75 to 80 nucleotides long.
• Picks up the appropriate amino acid floating in
the cytoplasm (amino acid activating enzyme)
• Transports amino acids to the mRNA.
• Have anticodons that are complementary to
mRNA codons.
• Recognizes the appropriate codons on the
mRNA and bonds to them with H-bonds.
 B. Transfer RNA (tRNA)
amino acid
attachment site methionine amino acid

U A C
anticodon
 tRNA Coupling
Aminoacyl-tRNA synthetase is required to catalyze the attachment of the
specific a.a. to it’s associated tRNA
 C. Ribosomal RNA (rRNA)
• Made up of rRNA is 100 to 3000 nucleotides
long.
• Important structural component of a ribosome.
• Associates with proteins to form ribosomes.
 Ribosomes
• Large and small subunits.
• Composed of rRNA (40%) and proteins (60%).
• Both units come together and help bind the
mRNA and tRNA.
• Two sites for tRNA
a. P site (first and last tRNA will attach)
b. A site
 Ribosomes

Large
subunit
P A
Site Site

mRNA
A U G C U A C U U C G

Small subunit
 Ribosomes
Protein formation requires orderly events to progress
Ribosomes possess binding sites to achieve this
mRNA binding site - holds mRNA strand in place
Aminoacyl-tRNA site (A-site) - holds one tRNA molecule
Peptidyl-tRNA site (P-site) - holds another tRNA molecule
Exit site (E-site) - holds a third tRNA molecule
 Translation
Nuclear
DNA membrane

Transcription
Pre-mRNA

Eukaryotic RNA Processing

Cell mRNA

Ribosome

Translation

Protein
 Translation
• Synthesis of proteins in the cytoplasm

• Involves the following:

1. mRNA (codons)
2. tRNA (anticodons)
3. rRNA
4. ribosomes
5. amino acids
 Translation
• Three parts:
1. initiation: start codon (AUG)
2. elongation:
3. termination:
stop codon (UAG)
 Translation

Large
subunit
P A
Site Site

mRNA
A U G C U A C U U C G

Small subunit
 Initiation

Initiation-requirements:

1. mRNA
2. Ribosome
3. Initiator tRNA (fMet tRNA in
prokaryotes)
4. 3 Initiation factors (IF1, IF2, IF3)
5. Mg2+
6. GTP (guanosine triphosphate)
Initiation-steps (e.g., prokaryotes):

1. 30S ribosome subunit + IFs/GTP bind to AUG start codon and

Shine-Dalgarno sequence composed of 8-12 purine-rich
nucleotides upstream (e.g., AGGAGG).

2. Shine-Dalgarno sequence is complementary to 3’ 16S rRNA.

3. Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All
new prokaryote proteins begin with fMet (later removed).

fMet = formylmethionine (Met modified by transformylase; AUG

at all other codon positions simply codes for Met)

mRNA 5’-AUG-3’ start codon

tRNA 3’-UAC-5’ anti-codon

4. IF3 is removed and recycled.

5. IF1 & IF2 are released and GTP is hydrolysed, catalyzing the
binding of 50S rRNA subunit.

6. Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

Initiation can be divided into four steps:
(1)dissociation of the ribosome into its 40S
and 60S subunits;
(2) binding of a ternary complex consisting
of met-tRNAi , GTP, and eIF-2 to the 40S
ribosome to form a preinitiation complex;
(3) bindingof mRNA to the 40S preinitiation
complex to form a 43S initiation complex;
(4) combination of the 43S initiation complex
with the 60S ribosomal subunit to form the
80S initiation complex.
 Initiation
aa1

1-tRNA
anticodon U A C
hydrogen A U G C U A C U U C G A
bonds codon mRNA
Elongation of a polypeptide:

1. Binding of the aminoacyl tRNA (charged tRNA)

to the ribosome.

2. Formation of the peptide bond.

3. Translocation of the ribosome to the next codon.

Binding of the aminoacyl tRNA to the
ribosome.
• Ribosomes have two sites, P site (5’) and A site (3’) relative to
the mRNA.

• Synthesis begins with fMet (prokaryotes) in the P site, and aa-

tRNA hydrogen bonded to the AUG initiation codon.

• Next codon to be translated (downstream) is in the A site.

• Incoming aminoacyl-tRNA (aa-tRNA) bound to elongation factor

EF-Tu + GTP binds to the A site.

• Hydrolysis of GTP releases EF-Tu, which is recycled.

• Another elongation factor, EF-Ts, removes GDP, and binds

another EF-Tu + GTP to the next aa-tRNA.

• Cycle repeats after peptide bond and translocation.

 Elongation
aa2
aa1

2-tRNA
1-tRNA
G A U
anticodon U A C
hydrogen A U G C U A C U U C G A
bonds codon mRNA
 Elongation

aa1 aa2

1-tRNA 2-tRNA
anticodon U A C G A U
hydrogen A U G C U A C U U C G A
bonds codon mRNA
Formation of the peptide bond.
• Two aminoacyl-tRNAs positioned in the ribosome, one in the P site
(5’) and another in the A site (3’).
• Bond is cleaved between amino acid and tRNA in the P site.
• Peptidyl transferase (catalytic RNA molecule - ribozyme) forms a
peptide bond between the free amino acid in the P site and
aminoacyl-tRNA in the A site.
• tRNA in the A site now has the growing polypeptide attached to it
(peptidyl-tRNA).

Fig. 6.16
 Elongation
peptide bond

aa1 aa2

1-tRNA 2-tRNA
anticodon U A C G A U
hydrogen A U G C U A C U U C G A
bonds codon mRNA
 Elongation
peptide bond
aa3
aa1 aa2

3-tRNA

1-tRNA 2-tRNA G A A
anticodon U A C G A U
hydrogen A U G C U A C U U C G A
bonds codon mRNA
 aa1 peptide bond
aa3
aa2

1-tRNA

U A C 3-tRNA
(leaves)
2-tRNA G A A

G A U
A U G C U A C U U C G A
mRNA

Ribosomes move over one codon

 peptide bonds
aa1 aa4

aa2 aa3

4-tRNA

2-tRNA 3-tRNA G C U

G A U G A A
A U G C U A C U U C G A A C U
mRNA
Translocation of the
ribosome to the next codon.
 peptide bonds
aa1 aa4
aa2

aa3

2-tRNA
4-tRNA
G A U
(leaves) 3-tRNA G C U

G A A
A U G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon

 peptide bonds aa5
aa1
aa2
aa4
aa3

5-tRNA

U G A
3-tRNA 4-tRNA

G A A G C U
G C U A C U U C G A A C U
mRNA
 aa1 peptide bonds aa5
aa2
aa3
aa4

5-tRNA

3-tRNA U G A
G A A 4-tRNA

G C U
G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon

 aa5
aa4 aa199 Termination
aa3 primary aa200
structure
aa2 of a protein

aa1
terminator
200-tRNA
or stop
codon
A C U C A U G U U U A G
mRNA
Termination of translation:
1. Signaled by a stop codon (UAA, UAG, UGA).
2. Stop codons have no corresponding tRNA.
3. Release factors (RFs) bind to stop codon and assist the
ribosome in terminating translation.
1. RF1 recognizes UAA and UAG
2. RF2 recognizes UAA and UGA
3. RF3 stimulates termination
4 termination events are triggered by release factors:
1. Peptidyl transferase (same enzyme that forms
peptide bond) releases polypeptide from the P site.
2. tRNA is released.
3. Ribosomal subunits and RF separate from mRNA.
4. fMet or Met usually is cleaved from the polypeptide.
 End Product
• The end products of protein synthesis is a
primary structure of a protein.

• A sequence of amino acid bonded together

by peptide bonds.
aa5
aa3 aa4
aa2 aa199

aa1 aa200
 Polyribosome
• Groups of ribosomes reading same mRNA
simultaneously producing many proteins
(polypeptides).

incoming
large
subunit

1 2 3 4 5 6 7
mRNA

incoming
small subunit polypeptide
 Question:
• The anticodon UAC belongs to a tRNA that
recognizes and binds to a particular amino
acid.

• What would be the DNA base code for this

amino acid?
 Answer:

• tRNA - UAC (anticodon)

• mRNA - AUG (codon)
• DNA - TAC
 PROTEOME
The proteome is the final
product of genome
expression and constitute
all the proteins present in
a cell at a particular time, It
is considered as the
central link between the
genome and the cell.
Protein Synthesis Inhibitor:

Many of the antibiotics utilized for the

treatment of bacterial infections as
well as certain toxins function
through the inhibition of translation.
Inhibition can be affected at all
stages of translation from initiation
to elongation to termination.
Protein Synthesis Inhibitor:
Several Antibiotic and Toxin inhibitors
of Translation:
Chloramphenicol: inhibits prokaryotic
peptidyl transferase
Cycloheximide: inhibits eukaryotic
peptidyl transferase
Diptheria toxin catalyzes ADP-ribosylation
of and inactivation of eEF-2
Erythromycin: inhibits prokaryotic
translocation through the ribosome
large subunit
 Protein Synthesis Inhibitor:
Fusidic acid: similar to erythromycin only by preventing
EF-G from dissociating from the large subunit
Neomycin: similar in activity to streptomycin
Puromycin: resembles an aminoacyl-tRNA, interferes
with peptide transfer resulting in premature
termination in both prokaryotes and eukaryotes
Ricin: found in castor beans, catalyzes cleavage of the
eukaryotic large subunit Rrna
Streptomycin: inhibits prokaryotic peptide chain
initiation, also induces mRNA misreading
Tetracycline: inhibits prokaryotic aminoacyl-tRNA
binding to the ribosome small subunit
 THE GENETIC CODE

Genetic code is degenerate, unambiguous,

none overlapping
The genetic code consists of 64 triplets of
nucleotides. These triplets are called codons.
Genetic code is required to account for all 20
amino acids found in proteins. A two-letter
code would have only 42 = 16 codons, which
is not enough to account for all 20 amino
acids, whereas a three-letter code would give
43 = 64 codons.
The 64 codons fall into groups, the members of
each group coding for the same amino acid.
 THE GENETIC CODE
Degeneracy all amino acid are coded by two,
three, four or six codons except tryptophan and
methionine have just a single codon each.
The code also has four punctuation codons, which
indicate the points within an mRNA where
translation of the nucleotide sequence should
start and finish.
The initiation codon is usually 5′-AUG-3′, which also
specifies methionine (so most newly synthesized
polypeptides start with methionine), with a few
mRNAs other codons such as 5′-GUG-3′ and 5′-
UUG-3′ are used.
The three termination codons are 5′-UAG-3′, 5′-
UAA-3′ and 5′-UGA-3′; these are sometimes
called amber, opal and ochre, respectively.
 THE GENETIC CODE

The code is not uambiguous because a given

codon designates only one amino acid.
One codon, AUG serves two related functions:
It signals the start of translation.
It codes for the incorporation of the amino acid
methionine (Met) into the growing polypeptide
chain.
The genetic code can be expressed as either
RNA codons or DNA codons:
 THE GENETIC CODE

RNA codons:
Occur in messenger RNA (mRNA) and
are the codons that are actually read
during the synthesis of polypeptides.
But each mRNA molecule acquires its
sequence of nucleotides by transcription
from the corresponding gene.
 THE GENETIC CODE

The DNA Codons: (genes at the level of

DNA):
These are the codons as they are read
on the sense (5' to 3') strand of DNA.
Except that the nucleotide thymidine
(T) is found in place of uridine (U),
they read the same as RNA codons.
However, mRNA is actually
synthesized using the antisense
strand of DNA (3' to 5') as the
template.
Semoga menjadi ilmu yang bermanfaat
Amin