Protein Synthesis

Protein Synthesis

• Genetic Information present within the

mRNA generates the linear order of amino
acids in proteins-process called translation
 Protein Synthesis
• The production (synthesis) of proteins.
proteins
3 phases:
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
 Various protein factors involved in protein synthe
Translation
Factors steps Functions
IF-1 Initiation Helps to stabilize 30S ribosomal subunit
Binds fmet-tRNA with 30S subunit mRNA
IF-2 Initiation complex; bind GTP and hydrolyse
IF-3 Initiation Binds 30S subunit with mRNA
Binds GTP; bring Aminoacyl-tRNA to A-site of
EF-TU Elongation ribosome
EF-TS Elongation Generates EF-TU
EF-G Elongation Helps in translocation of ribosome
Helps to dissociate polypeptide from tRNA
RF-1 Termination ribosome complex; specific for UAA and UAG
Helps to dissociates polypeptide; specific for
RF-2 Termination UGA and UAA
RF-3 Termination Stimulates RF-1 and RF-2
 Chain Initiation in Prokaryotes
(E.Coli)

• In E.coli, the initiation process involves,

-a special initiator tRNA,
-30s subunit of ribosome,
-an mRNA molecule,
-three soluble protein initiation factors
IF-1, IF-2, IF-3 and
-one molecule of GTP.
• Translation occurs on 70 s Ribosome (after translation they
disassociates in to 30 and 50 s subunits).

• The synthesis of polypeptide is initiated by a special tRNA,

designated as tRNAfmet, in response to a translation initiation
codon (usually AUG).

• The methionine on the initiator tRNAfmet has the amino group

blocked with a formyl group
 Chain Initiation in Prokaryotes (E.Coli)
• All polypeptides begin with methionine during
protein synthesis.

• Amino terminal methionine is subsequently cleaved

from many polypeptides.

• A distinct methionine tRNA, tRNAmet responds to the

internal methionine codons.
amino acid
attachment site methionine

Both methionine tRNAs

have the same anticodon, and
Both responds to same codon
(AUG)
U A C

anticodon
 a special initiator tRNA
• Only methionyl-tRNAfmet interacts with protein initiation factor IF-2 to
begin initiation.

• Polypeptide chain initiation begins with the formation of two complexes

-Initiation factor IF-2 and methionyl-tRNA f met
-Complex-1
-mRNA + 30 s ribosomal subunit + IF3
-Complex-2
• The 30s subunit /mRNA complex will form only in the presence of IF3.

• The formation of the 30s subunit /mRNA complex depends on base-

paring between a nucleotide sequence near the 3’end of the 16s rRNA
and a sequence near 5’end of the mRNA molecule.
 Shine-Dalgarno Sequence
• Prokaryotic mRNA contains a conserved
polypurine tract, consensus of “AGGAGG”,
located about seven to eight bases upstream from
the AUG initiation codon.

• The conserved hexamer, called Shine-Dalgarno

sequence after the scientist who discovered it.

• Eukaryotes possess a Kozak sequence

“A/GCCACCAUGG”, which lies within a short 5'
untranslated region, directs translation of mRNA
 Shine-Dalgarno Sequence

Translation
Shine-Dalgarno
initiation codon
sequence

mRNA 5’ AACACAGGAGGAUUAUCCAUGUCG
16SRNA 3’ UCCUCCUAAUAGGUACAGC

Region of base pairing

 30 s initiation complex
• The IF-2 / methionyl-tRNAfmet complex and mRNA
+30 s ribosomal subunit+IF3 complex subsequently
combines with each other with the help of IF-1 and
GTP resulting 30 s initiation complex.

• The final step in the initiation is addition of 50s

subunit to 30 s initiation complex to produce the
complete 70 s ribosome.
 Final step in the initiation
• The addition of 50 s subunit to the complex,
positions the initiator tRNA, methionyl-tRNA f met in
peptidyl (P) site with anticodon of tRNA aligned
with AUG initiation codon of the mRNA.

• methionyl-tRNAfmet is the only aminoacyl tRNA that

can enter the P Site directly without first passing
through the aminoacyl (A)site.
 Chain Elongation
• With the initiator positioned in the P site, the
second codon of the mRNA is in register with A
site and set the stage for second phase in
polypeptide synthesis, chain elongation.

• At the beginning of the elongation cycle, the

peptidyl site is filled with methionyl-tRNA f met , and
aminoacyl site are empty.
 Aminoacyl-tRNAs are Delivered to the A Site by

Elongation Factor EF-Tu

Step-1
• Aminoacyl-tRNAs do not bind to the ribosome on
their own. Instead, they are "escorted" to the
ribosome by the elongation factor EF-Tu.

• Once a tRNA is aminocylated, EF-Tu binds to the

tRNA's 3' end, masking the coupled amino acid.

• This interaction prevents the bound aminoacyl-tRNA

from participating in peptide bond formation until it is
released from EF-Tu.
EF-Tu escorts aminoacyl-tRNA to the
A site of the ribosome. Charged tRNAs
are bound to EF- Tu-GTP as they first
interact with the A site of the ribosome.
When the correct codon-anticodon
interaction occurs, EF-Tu interacts
with the factor binding center,
hydrolyzes its bound GTP and is
released from the tRNA and the
ribosome.
 The Ribosome Uses Multiple Mechanisms to Select
against Incorrect Aminoacyl-tRNAs

• The error rate of translation is between 10 -3 to 10-4.

• That is, no more than I in every 1,000 amino acids

incorporated into protein is incorrect.

• The ultimate basis for the selection of the correct

aminoacyl-tRNA is the base pairing between the
charged tRNA and the codon displayed in the A site
of the ribosome.
Correct base-pairing allows EF-Tu
bound to the aminoacyl-tRNA to
interact with the factor binding
center inducing GTP hydrolysis
and EF-Tu release.
Only correctly base paired
aminoacyl-tRNAs remain
associated with the
ribosome as they rotate
into the correct position
for peptide bond
formation.

This rotation is referred to

as tRNA accommodation.
 Release Factors Terminate Translation in
Response to Stop Codons
• The ribosome's cycle of aminoacyl-tRNA binding, peptide
bond formarion, and translocation continues until one of
the three stop codons enters the A site.

• It was initially postulated that there would be one or more

chain-terminating tRNAs that would recognize these
codons.

• However, this is not the case. Instead, stop codons are

recognized by proteins called release factors (RFs) that
activate the hydrolysis of the polypeptide from the peptidyl-
tRNA.
 Release factors RF1 and RF2.

• Prokaryotes have two class I release factors

called RF1 and RF2.

• RF1 recognizes the stop codon UAG, and RF2

recognizes the stop codon UGA.

• The third stop codon, UAA, is recognized by both

RF1 and RF2.
 Ribosomes

Large
subunit
P A
Site Site

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

Small subunit
 Translation

Large
subunit
P A
Site Site

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

Small subunit
 Initiation
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 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
 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
 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
 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
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
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
 Comparisons between prokaryotic and
eukaryotic protein synthesis

• One big difference is that prokaryotes can transcribe and

translate the same gene simultaneously.
• The new protein quickly diffuses to its operating site.
• In eukaryotes, the nuclear envelope segregates
transcription from translation.
• In addition, extensive RNA processing is inserted between
these processes.
– This provides additional steps whose regulation helps
coordinate the elaborate activities of a eukaryotic cell.
• In prokaryotes
– There are a number of ribosomes attached to a single mRNA, all
at different stages
– Translation of mRNA can begin before transcription is complete
because mRNA is being made in the cytoplasm

• In eukaryotes
– Transcription occurs in the nucleus
– mRNA must move through the membrane into the cytoplasm
– mRNA undergoes processing before it leaves the nucleus
• Eukaryotic genes are made of exons, regions of DNA
expressed and introns, intervening regions that do not
encode proteins
Eukaryotic translation
 Cap-dependent initiation
Initiation of translation usually involves the interaction of
certain key proteins with a special tag bound to the 5'-end of
an mRNA molecule, the 5' cap.

The protein factors bind the small ribosomal subunit (also

referred to as the 40S subunit), and these initiation
factors hold the mRNA in place. The eukaryotic Initiation
Factor 3 (eIF3) is associated with the small ribosomal subunit,
and plays a role in keeping the large ribosomal subunit from
prematurely binding. eIF3 also interacts with the eIF4F
complex which consists of three other initiation factors:
eIF4A,eIF4E and eIF4G. eIF4G is a scaffolding protein which
directly associates with both eIF3 and the other two
components. eIF4E is the cap-binding protein.
It is the rate-limiting step of cap-dependent initiation, and is
often cleaved from the complex by some viral proteases to
limit the cell's ability to translate its own transcripts.

This is a method of hijacking the host machinery in favor of

the viral (cap-independent) messages. eIF4A is an ATP-
dependent RNA helicase, which aids the ribosome in
resolving certain secondary structures formed by the mRNA
transcript.
There is another protein associated with the eIF4F complex
called the Poly(A)-binding protein(PABP), which binds
the poly-A tail of most eukaryotic mRNA molecules. This
protein has been implicated in playing a role in circularization
of the mRNA during translation. pre-initiation complex (43S
subunit, or the 40S and tRNA) accompanied by the protein
factors move along the mRNA chain towards its 3'-end,
scanning for the 'start' codon (typically AUG) on the mRNA,
which indicates where the mRNA will begin coding for the
protein.
In eukaryotes and archaea, the amino acid encoded by the
start codon is methionine. The initiator tRNA charged with
Met forms part of the ribosomal complex and thus all
proteins start with this amino acid (unless it is cleaved away
by a protease in subsequent modifications).

The Met-charged initiator tRNA is brought to the P-site of

the small ribosomal subunit by eukaryotic Initiation Factor 2
(eIF2). It hydrolyzes GTP, and signals for the dissociation of
several factors from the small ribosomal subunit which
results in the association of the large subunit (or the 60S
subunit). The complete ribosome (80S) then commences
translation elongation, during which the sequence between
the 'start' and 'stop' codons is translated from mRNA into an
amino acid sequence—thus a protein is synthesized.
Regulation of protein synthesis is dependent on
phosphorylation of initiation factor eIF2 which is a part of the
met-tRNAi complex.

When large numbers of eIF2 are phosphorylated, protein

synthesis is inhibited. This would occur if there is amino acid
starvation or there has been a virus infection.

However, naturally a small percentage is of this initiation

factor is phosphorylated. Another regulator is 4EBP which
binds to the initiation factor eIF4E found on the 5’ cap on
mRNA stopping protein synthesis. To oppose the effects of
the 4EBP growth factors phosphorylate 4EBP reducing its
affinity for eIF4E and permitting protein synthesis.
 T C G TTC A A A
AG T T
Template
C AAGT
AA1
Strand
Nucleus

AGC
AA1 •AA2 AA3
Cytoplasm

AAG U U U
U C G UU C A A A
 T C G TTC A A A
AG T T
template
C AAGT
Strand
U C G UU C A A A
mRNA
 T C G TTC A A A
AG T T
template
C AAGT
Strand
Nucleus U C G UU C A A A
mRNA

Cytoplasm

Ribosome
 T C G TTC A A A
AG T T
Template
C AAGT
Strand
Nucleus U C G UU C A A A
mRNA

Cytoplasm

U C G UU C A A A
 T C G TTC A A A
AG T T
Template
C AAGT
Strand
Nucleus

AA
1 Cytoplasm

GC
A U CG
A’s UU C A A A
N
tR
 T C G TTC A A A
AG T T
template
C AAGT
Strand
Nucleus

AA1
Cytoplasm
tRNA’s
AGC
U C G UU C A A A
 T C G TTC A A A
AG T T
template
C AAGT
Strand
Nucleus
ATP

AA1 •AA2
Cytoplasm
tRNA’s
AGC AAG
U C G UU C A A A
 T C G TTC A A A
AG T T
Template
C AAGT
AA1
Strand
Nucleus
ATP

AA1 •AA2 AA3

Cytoplasm
AG
C AAG U U U
U C G UU C A A A
 T C G TTC A A A
AG T T
template
C AAGT
AA1
Strand
Nucleus

AGC
AA1 •AA2 AA3
Cytoplasm

AAG U U U
U C G UU C A A A
 T C G TTC A A A
AG T T
Template
C AAGT
Strand
AA1
Nucleus

AGC
AA1 •AA2 AA3
Cytoplasm

AA UUU
G
U C G UU C A A A
 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
 Attachment of Amino acid to tRNA

anticodon loop
Extending from the acceptor
stem, the 3' end of each tRNA
has the sequence CCA.
acceptor
tRNA stem
anticodon

The appropriate amino acid

is attached to the ribose of Phe
the terminal adenosine (A, in tRNA

red) at the 3' end.

acceptor
stem
 O O O O
H
R C C O 
O P O P O P O CH 2 Adenine
O
NH3+ O O O H H
Amino acid ATP H H
 OH OH

O O
H
R C C O P O CH2 Adenine
O
NH2 O H H  PPi
Aminoacyl-AMP H H
OH OH

Aminoacyl-tRNA Synthetases catalyze linkage of the

appropriate amino acid to each tRNA.
The reaction occurs in 2 steps.
In step 1, an O atom of the amino acid a-carboxyl attacks the P
atom of the initial phosphate of ATP.
 O O
H
R C C O P O CH2 Adenine
O
NH2 O H H

Aminoacyl-AMP H H
OH OH
tRNA
In step 2, the 
tRNA AMP
2' or 3' OH of
O
the terminal
Adenine
adenosine of O P O CH2
O
tRNA attacks (terminal 3’nucleotide
O H H
of appropriate tRNA)
the amino H 3’
O
2’H
OH
acid carbonyl
C O
C atom. Aminoacyl-tRNA
HC R

NH3+
 Aminoacyl-tRNA Synthetase - summary

1. amino acid + ATP  aminoacyl-AMP + PPi

2. aminoacyl-AMP + tRNA  aminoacyl-tRNA + AMP
The 2-step reaction is spontaneous overall, because the concentration of
PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.
There is generally a different Aminoacyl-tRNA Synthetase (aaRS) for each
amino acid.

Accurate translation of the genetic code depends on attachment of each

amino acid to an appropriate tRNA.

Each aaRS recognizes its particular amino acid & tRNAs coding for that
amino acid.

Identity elements: tRNA domains recognized by an aaRS.

 Codon-tRNA Interactions

• Translation of a sequence of nucleotides in

mRNA into the correct sequence amino
acids requires the accurate recognition of
codon by aminoacyl-tRNAs.
Genetic Code
 The Genetic Code

• Genes direct the synthesis of proteins

• Nucleotides (A T G & C) control the sequence of

20 amino acids present in proteins

• Cracking the code was one of the most exciting

events in the history of science
 The most important features of
Genetic Code
• Genetic code is composed of Nucleotide triplets.

• Three nucleotides in mRNA specify one amino acid

in the polypeptide chain.

• The genetic code is non overlapping, in rare cases

genes overlap and nucleotide sequence is read in
two different reading frames.
 The most important features of
Genetic Code

• The genetic code is comma-free

• During translation codons are read consecutively

• The genetic code contains start and stop codons

• The genetic code is nearly universal

 Three nucleotides per codon
• Twenty different amino acids are incorporated into
polypeptide chain during translation.

• Thus, at least 20 different codons must be formed with

the four bases available in mRNA.

• Two bases per codon results 42; so 16 codons, 20 amino

acids, clearly not enough.

• Three bases per codon results 43 ; so 64 codons

Initiation and termination codons

• In both pro and eukaryotes, the codon AUG is

used to initiate polypeptide chain initiation.

• In eukaryotes, the start codon must be at the

5’ end of the mRNA.

• Three codons UAG, UAA and UGA specify

polypeptide chain termination.
 A degenerate and ordered code
• All amino acids except Methionine and Tryptophan are
specified by more than one codon

• Three amino acids Leucine, Serine and Arginine each one

is specified by six different codons.

• Isoleucine has three codons.

• Other amino acids each has either two or four codons

• The occurrence of more than codon per amino acid is

called as Degeneracy
 A degenerate and ordered code
• The degeneracy in the genetic code is not
random, instead it is highly ordered.

• In most cases, the multiple codons specifying a

given amino acid differ by only one base, the
third or 3’ base of the codon.

• The genetic code degeneracy is of two types

1. Partial degeneracy
2. Complete degeneracy
 1. Partial degeneracy

• Partial degeneracy occurs when the third base

may be either of two pyrimidines (U or C) OR
Alternatively, either of two purines (A or G)

Changing the third base from a Purine to

Pyrimidine or Pyrimidine to Purine will change the
amino acid specified by the codon.
E.g. CUU and CUC code for leucine.
 2. Complete degeneracy

• Any of the four bases may be present at the third position

in the codon, and the codon will still specify the amino acid.

Example: Valine is coded by

GUC
GUU
GUA
GUG
 Genetic code is nearly Universal
• Information from many species indicate that the genetic code
is nearly universal, that the codon have same meaning.

Most important exceptions are seen in Mitochondria of

mammals and yeast

– UGA specifies Tryptophan rather than chain termination.

– AGA and AGG are chain termination codons, rather than

Arginine
 Recognition of codons by t-RNAs
THE WOBBLE HYPOTHESIS

• The genetic code is read during translation via

adapter molecules, tRNAs, that have 3-base
anticodons complementary to codons in mRNA.

• The hydrogen bonding between the bases of

codon and anti codon follow strict base pairing
rules for the first two bases only.
 THE WOBBLE HYPOTHESIS

• The base pairing involving the third base of the

codon is less stringent- Crick called Wobble to
this site.

• Crick proposed that the Wobble would allow

several types of base-paring at the third codon
base in the codon-anticodon interactions.
 THE WOBBLE HYPOTHESIS
Base in anti-Codon Base in Codon

G U or C
C G
A U
U A or G
I A, U or C

Base pairing between the 5’ base of the anticodon of tRNA and 3’

base of codon (mRNA) according to Wobble Hypothesis
Translational control
Important steps in translation

Ribosome consists of 3 sites, A-site, P-site and Exit site

Initiator tRNA binds to the P-site

Chain elongation: Requires elongation factors. These are EF-Tu, EF-Ts and EF-G in
prokaryotes and EF-1 and EF-2 in eukaryotes

The second amino acid-tRNA complex (aa2-tRNA) now occupies the A site.

Formation of peptide bod takes place by transfer of fMet to the second amino acid
(aa2). The catalyzing enzyme is peptidyl transferase.

Translocation: The aa2-tRNA complex moves from the A site to the P-site which is
called translocation.

Translocation involves movement of the ribosome relative to mRNA in the 5’- 3’

direction.

Note: The tRNA molecule of fMet is unloaded from the ribosome. The third amino acid-
tRNA complex (aa3-tRNA) now occupies the vacant P site.

Chain termination: chain elongation continues until a termination codon (UAA, UAG or
UGA) reaches the ribosome. The chain is then terminated and released from the
ribosome. This process requires RF factor in eukaryotes
Difference between prokaryotic and eukaryotic translation
Prokaryotes Eukaryotes
Activating enzymes Aminoacyl tRNA Aminoacyl tRNA
Synthetases Synthetases

mRNA Often polycistronic Monocistronic

Initiator t RNA tRNAfMet tRNAiMet
Initiation amino acid N-formylmethionine (f Methionince (Met)
Met)

Initiation factors IF1,2,3 eIF1,1A,2,2B,3,4F

etc
Elongation factors EF-T (EF-Tu, Ts, G) EF-1 and 2
Termination codon UAA/UAG/UGA UAA/UAG/UGA
Release factors RF-1,2,3 RF+GTP
Regulation of Protein
Translation
 Why Regulate Translation?
Translation: Produces proteins rapidly
Produces proteins locally
Produces single proteins or classes of
proteins
BUT, It is very costly in energy.
 There are two overall mechanisms of
translation control

1. Regulation by modifying proteins

2. Regulation using micro RNAs (miRNA’s)

MicroRNAs are 21-nucleotide-long regulators

of gene expression that gain access to
their target mRNAs by complementary
base pairing.