Protein Synthesis :

The decoding of information in mRNA sequences to

amino acid sequences of a protein by cellular
translational machinery

Machinery:
1.Ribosomes: 70 S prokaryotic and 80S eukaryotic

2. Soluble factors/ enzymes like amino acyl

synthetases , protein factors involved in
initiation elongation and termination

3. Transfer RNAs

4. Messenger RNA
Secondary Structure: clover leaf L- shaped three dimensional struc
Prokaryotic Eukaryotic
tRNAf-met initiator tRNA tRNAiMet- initiator tRNA

Start Methionine is formylated Start methionine is unformylated

IF2 delivers initiator tRNA to 30S eIF2/eIF2B delivers initiator tRNA to 40S
ribosome

Initiation requires three factors Initiation step requires a dozen factors:

IF1, IF2 and IF3 eIF1, 1A, eIF2/2B, eIF5, eIF5B, eIF6, eIF3,
eIF4s (A, B, E and G)

One GTP is hydrolyzed in initiation Two GTPs are hydrolyzed in initiation step

Formation of 70S complex Formation of 80S initiation complex

Shine Dalgarno sequence is present No Shine Dalgarno like sequence

mRNA is not circularized mRNA can be pesudocircularized due to

interaction
between proteins bound to 5 and 3
ends of mRNA
Peptide bond is catalyzed by 23S rRNA Peptide bond is catalyzed by 28S rRNA.

Elongation requires EFTu/TS and EF-G Elongation requires eEF1 (, and, eEF-2

Termination requires RF1 and RF2 Termination requires eRF1

For example, if the G3: U70 nucleotides of tRNAala are used to
replace the 3:70 base pair of tRNAcys or tRNAphe, then these
modified tRNAs are recognized by alanyl tRNA synthestase and
charged with alanine suggesting that G3:U70 base pair is a critical
identity element in tRNAala for its specific synthetaseG2

Amino acyl synthetases recognize

specific tRNA sequences

5 end
(AA stem) D Loop Anticodon Loop Amino acid acceptor Stem
3

G3 U70
tRNA Ala

G20 G34 A35 A36 A73-----

tRNA charging involves two steps:
a)Step 1, adenylation of amino acid
that occurs in the presence of ATP
where pyrophosphate is liberated

b) In the second step, 3 OH group

(end of A of CCA) of a transfer RNA
joins the adenylated amino acid ,
liberating AMP. tRNA joins the amino
acid by a high energy bond.

c) The process is mediated by an

enzyme called amino acyl tRNA
synthetase.
 EUKARYOTIC RIBOSOME

Subunits : 40S + 60S = 80S monosome

Polysomes- mRNA with one 80S, two 80S
Three 80S, 4 80S monosomes 2n, 3n, 4n

PROKARYOTIC RIBOSOME

Subunits : 30S + 50S = 70S monosome

Polysomes- mRNA with one 70S, two 70S
Three 70S, four 70S monosomes; 2n, 3n, 4n
Aminoacylated initiator tRNA is formylated by methionyltRNA
transformylase (MTF). The enzyme catalyzes the transfer of a formyl
group from N10-formyltetrahydrofolate to the -amino group of the
methionine of Met-tRNAf Met. The most important determinant for
formylation of the initiator tRNA is the absence of a 1:72 base pair

Methionine
tRNAfMet + amino acyl
synthetase
Met-tRNAfMet
formate from N10 tetra
hydofolate
N-formyl-Met-tRNAfMet
Protein Synthesis
N-formyl Met-aa1- aa2---- aan
Formate
Met-aa1-aa2-----aan
Methionine (Metspecific
peptidase)
Aa1-aa2----aan finished protein
 Initiation factors and their functions :
Prokaryotes (E.coli) Three initiation factors IF1, IF2 and IF3 help to
position the initiator tRNA in the P site of ribosome on the start AUG codon
in mRNA.
IF3 bound 30S subunit cannot join the 50S subunit to form 70S initiation
complex unless the factor is released. IF3 prevents association of the
ribosome subunits. IF3 especially stabilizes the 30S initiation complex.

b) It also promotes the dissociation of non initiator aminoacylated

tRNAs binding to the P-site of 30S ribosomal subunit,

c) the dissociation of 70S complexes, and in the recycling of

ribosomal subunits at the end of synthesis.

IF1, the smallest protein factor among all the three initiation factors (8.2
kDa in E. coli). It promotes the interaction between IF2 and 30S
subunit and more specifically the interaction of IF2. fMet. tRNA.GTP
with the initiation codon of the mRNA in the P-site
IF2 is the largest initiation factor (approx 89-90 kDa)

It has highest affinity to ribosomes compared to other factors and

joins fMe.tRNAfMet

The complex IF2.GTP will promote the association of 30S

initiation complex with 50S subunit to form 70S initiation
complex.

Hydrolysis of GTP bound to IF2 by a GTPase occurs in the presence of

ribosomes.

The GTP hydrolysis however at the end of initiation facilitates

a) the release of IF2.GDP from the 30S initiation complex, and
b) the adjustment of initiator tRNA in the P-site of ribosome
with proper codon-anticodon pairing.

GTP hydrolysis by a GTPase however occurs only after the subunits

are joined.

Hydrolysis of the GTP may cause a conformational change in

ribosome that would facilitate the release of all the factors. IF2.GDP
1.Right tRNA is required to accept the right amino acid. Sequence of tRNA contains the
necessary information.

2.The joining of amino acid with the tRNA requires ATP and aminoacyl synthetase enzyme.

3.Two types of tRNAs: initiator and elongator tRNAs

4.Only one initiator tRNA is present in any system, elongator tRNAs however are many.

5.Initiator tRNA s are charged with methionine--- which is mostly the start or first amino acid to
be incorporated. Rarely Valine GUG can serve as the initiating aa.

6. A methionine or other amino acids can also be incorporated in the middle of a polypeptide. It
would be brought by respective elongator tRNA. EX: tRNAmMET (for met) .

7. In prokaryotes, the initiator tRNA is called tRNAfMet where as in eukaryotes, it is called

tRNAiMet. The difference is because the prokaryotic tRNA recognizes methionine that is
formylated subsequently . Similar formylation does not occur in eukaryotes.

8. Finally, initiator tRNAs after combining with the amino acid methionine, deliver them to 30S
ribosomal subunits in prokaryotes or to 40S ribosomal subunits in eukaryotes

9. The delivery of initiator RNA to the respective ribosomal subunits require IF2 in prokaryotes
and eIF2/ eIF2B in eukaryotes. The elongator tRNAs deliver their amino acids with the help of
EF.TU.TS in prokaryotes and eEF1 (with a and bg subunits) in eukaryotes.
 Initiation of Protein Synthesis in Prokaryotes; Initiator and el
FIG. Initiator and elongator methionine-accepting
tRNAs. Cloverleaf representation of methionine-
accepting tRNAs: (A) initiator tRNA and (B)
elongator tRNA. The regions important for initiator
tRNA identity are highlighted. Details are given in
the text.
The significant features include
(i) absence of a Watson-Crick
base pair between positions 1
and 72 in the acceptor stem,
(ii) three conserved consecutive
GC base pairs in the anticodon
stem, and
(iii) the presence of a purine-11
pyrimidine-24 in contrast to the
pyrimidine-11purine-24 base
pair found in other tRNAs .
The GC pairs make the
anticodon loop less flexible
compared to the anticodon loop
in elongator tRNAs and are
important for targeting the
initiator tRNA to the ribosomal P-
site.
 Start site selection (AUG) in pro and eukaryotes:
Shine Dalgarno Sequence or Ribosome Binding Site ( RBS)

Polypurine rich
sequence in mRNA
base pairs with
polypyrimidine rich
sequence
In the 16S rRNA in
bacteria which
contains polycistronic
mRNA (panel a) Panel b represents monocistronic mRNA in eukaryotes.
No shine dalgarno sequence. G/A preceding start site
are imp
A- amino acylated tRNA binding site
P-Peptidyl tRNA binding site
E-Exit site , deacylated tRNA
The tRNA and mRNA can
bind in either order and
independently of each other.
The three steps in Elongation:

(i)delivery of the amino acylated tRNA to the A

site of 70S ribosome complex;

(ii) peptide bond formation between adjacent

amino acids by petidyl transferase (ribosomal
RNA or ribozyme); and

(iii) the movement of mRNA by three

nucleotides
(i) delivery of the amino acylated tRNA to the A site of 70S ribosome
complex- EF Tu/Ts catalyzes the delivery

TS + GTP
 EF.Tu/Ts factor promotes the joining of amino acylated tRNA to the
A-site in ribosome in elongation
Elongation factor, EF.Tu.GTP complex catalyzes the attachment of an
aminoacylated tRNA (aatRNA) to the A site in 70S ribosome

Unlike IF2, EF.Tu has a high affinity for GDP. However GDP inhibits the joining of
aminoacylated tRNA to EF.Tu. Exchange of GTP for the bound GDP is critical.

This GDP/GTP exchange is catalyzed by a guanine nucleotide exchange factor

called EF.Ts. EF. Tu. GDP + Ts >>>> GTP>>> EF. Ts.GTP + aminoacylated
tRNA>> EF.Tu.GTP.aa tRNA

EF.Tu binds the 3end of tRNAs and protects the attached amino acid from entering
into the peptidyl site. After the delivery of aminoacylated tRNA into the A site of
ribosome, the GTP bound to EF.Tu is hydrolyzed and the EF.Tu.GDP is released.

EF.TU is recycled by EF.Ts and GTP to form EF.Tu.GTP that is competent to join
aminoacylated tRNA. The GTPase activity that hydrolyzes the GTP bound to EF.Tu
is stimulated when the ternary complex, EF.Tu.GTP. aa tRNA, joins the A site of
the ribosome and interacts with the factor binding center of the ribosome.

Efficiency of GTPase activity is high and dependent on the correct base pairing
between codon-anticodon. It acts as one of the mechanisms to ensure proper
codon-anticodon interactions.
Peptide bond formation occurs between amino acid present at the 3 end of tRNA of
the growing polypeptide chain in P site and the aminoacylated tRNA present in the A
site.

The peptide bond formation requires N-terminus to be synthesized before the C-

terminus.

The growing polypeptide attached to the peptidyl tRNA is transferred to the tRNA
present in the A-site (contains a newly arrived amino acylated tRNA)
Peptide bond formation takes place Carboxyl
without the hydrolysis of any NTP. group of aa is
attached to 3
The alpha-amino group of the aminoacyl- end of amino
tRNA attacks the carbonyl group of the acylated
growing polypeptide attached to peptidyl- tRNA
tRNA

Peptide bond formation is driven by

breaking high energy acyl bond that joins
growing polypeptide chain to tRNA.

The high energy acyl bond was created

during tRNA charging by amino acyl
synthetase in the presence of ATP
 Head and Tail Growth
In head growth the head of the growing polymer is "activated"
it carries the energy for the addition of the next monomer. This
"activation" energy is depicted below as a red bond. Each of the
incoming monomers is also "activated" but the energy of the
activated bond will be used for the next addition once the
monomer is added to the growing polymer.

The classic example of head growth is protein synthesis.

Fatty acids are also made in this way.

Ex : Protein Ex: nucleic acid

synthesis synthesis

In tail growth, the head of the growing polymer is not activated. The energy for the
addition of each monomer is supplied by the incoming activated monomer.
Protein Synthesis and Fatty acid SynthesisDNA synthesis and Glycogen synthesi

If DNA synthesis utilized a head growth

mechanism, then proofreading would not
have evolved since removal of the last
monomer also removes the activated
head of the growing chain. That's why
there's no proofreading in protein
The synthesis of storage carbohydrates such as starch and glycogen
doesn't involve proofreading but there's still a very good reason why the
mechanism is tail growth.

Recall that starch and glycogen are polymers of glucose and their role is
to store glucose as a potential carbon source in time of need. When the
need arises, the ends of the polysaccharide chains are nibbled back
releasing glucose molecules (as glucose-6-phosphate). These
molecules enter the glycolysis pathway

The degradation reaction terminates when the immediate need for

glucose has been met. Later on, in time of plenty, the starch and
glycogen chains can be re-extended by adding more glucose residues.
The reason why this is possible is because starch and glycogen
synthesis is an example of tail growth just like nucleic acid synthesis. If
nibbling the ends of the polysaccaride chains removed the activated
head, as it would in the case of head growth, then the synthesis
reaction could not occur. Thus, the fundamental reason why tail growth
evolved in both nucleic acid synthesis and glycogen synthesis is the
same.
 Peptide bond formation between adjacent amino acids by
petidyl transferase (ribosomal RNA or ribozyme)
The Ribosome Is a Ribozyme
Once the correctly charged tRNA has been placed in the A site and has
rotated into the peptidyl transferase center, peptide bond formation takes
place.

This reaction is catalyzed by RNA, specifically the 23S rRNA component

of the large subunit.

Early evidence for this came from experiments in which it was shown that
a large subunit that had been largely stripped of its proteins was still able
to carry out peptide bond formation. Proof that the peptidyl transferase is
entirely composed of RNA has come from the high-resolution, three-
dimensional structure of the ribosome, which reveals that no amino acid
is located closer than 18 A0 from the active site. Because catalysis
requires distances in the 1 - 3 A0 range, it is clear that the peptidyl
transferase center is a ribozyme. That is an enzyme composed of RNA

Peptide bond between adjacent amino acids is catalyzed by an RNA

or RNA enzyme i.e., 23S rRNA of the large ribosomal subunit but not
by a protein. Hence the large ribosomal subunit (50S or 60S) is
called the catalytic center or subunit
Translocation of tRNA and mRNA are promoted by EF-
G.GTP. Formation of peptide bond leaves the tRNA
deacylated in the Psite and the growing polypeptide chain in
the A site. Then the mRNA moves by three nucleotides to
bring in the next codon.

The deacylated tRNA moves to the E site whereas the

growing polypeptide chain located at this point in A site moves
to the P site thus allowing room for a new amino acylated
tRNA to enter the A site.

The evacuation of deacylated tRNA from the E-site and the

movement of mRNA by one codon (three bases) relative
to the ribosome requires the mediation of elongation
factor-G (EF-G) and GTP.
EF-G. GTP acts like a translocase enzyme and drives the
translocation of tRNA and mRNA. Translocation step requires the
hydrolysis GTP bound to EF-G. Hydrolysis of GTP is stimulated when
EF-G contacts the factor-binding center of the ribosome.

Interestingly, EF.G.GDP structure appears to resemble

EF.Tu.GTP.aa.tRNA complex which also binds to the A-site. This is a
kind of molecular mimicry

GTP hydrolysis triggers a conformational change in the ribosome that

facilitates the translocation of the growing polypeptide chain from A site to
P-site and also formation of EF-G.GDP.

This movement of mRNA during translocation has been further supported

by the fact that movement of rare frame shift tRNAs that have four
nucleotides in their anticodon region can move the mRNA by four bases.
 Summary of Initiation and Elongation Steps in Protein Synthesis
Initiation highlights: Initiator tRNA charging with the amino acid, methionine
(formyl methionyl tRNA or initiator tRNA in prokaryotes and eukaryotes
respectively). Charging requires 2 high energy bonds of ATP; Charged initiator
tRNA is delivered to 30S or 40S ribosomes and is mediated by IF2 (a monomeric
protein) or eIF2 ( a heterotrimer with three subunits (and ) in prokaryotes and
eukaryotes respectively.

1. The joining of initiator tRNA to IF2 or eIF2 is mediated by GTP and GDP inhibits
the joining. 2. The bound GTP is hydrolyzed when the large subunit joins the small
subunit carrying the initiator tRNA and mRNA to form 70S or 80S initiation
complex . 3 initiator factors IF1, IF2 and IF3 play important roles in initiation of
prokaryotes, where as a dozen protein factors are required in the initiation step of
eukaryotes.

Initiator tRNA is positioned in the P (peptidyl) site of ribosome while the A site and E
site are empty tRNAs, that bring in amino acids based on the sequence
information in mRNA, after the initiation step , are all called elongator tRNAs.

Elongator tRNAs are amino acylated (charged with respective amino acids) and
are delivered to the A site of 70S or 80S initiation complexes by EF.Tu.Ts or eEF1
( with and -subunits). EF.Tu, like eukaryotic eIF2, has a higher affinity for GDP.
The GDP is exchanged on EF.Tu for GTP by GDP/GTP exchange factor called Ts
where as eIF2B exchanges GDP for GTP on eIF2. Elongation steps include
Delivery of aminoacylated to tRNA to 30S/40S and to
70S/80S : What factors are required?
(Prokaryotes/Eukaryotes)
What are aminoacylated tRNAs? Initiator or elongator tRNAs
charged with respective amino acids)
Prokaryotes:
Initiation : IF2.GTP + Met tRNAf ( initiator tRNA carrying
formylated methionine) >> 30S

Elongation: EF.TU.GTP. Aminoacylated tRNA > 80S (Ts is

exchanges GDP bound to EF-Tu for GTP >> 70S

Eukaryotes:
Initiation: eIF2. GTP. Met.tRNAi ( initiator tRNA carrying
methione) >> 40S. eIF2B is required for the exchange of GDP
bound to eIF2 for GTP.

Elongation: eEF1 .GTP. Amino acylated tRNA (or elongator

 Protein Synthesis contd: Termination

Release Factors (RFs) Terminate Translation in Response

to Stop Codons

Stop codons, UAA, UAG, UGA, are not recognized by any

tRNAs, but are recognized by proteins called release
factors (RFs).

The presence of stop codons in the A site of ribosome

signal the binding and recognition of stop codons by
release factors called RFs.

RFs activate hydrolysis of the ester bond between the 3

nucleotide of tRNA located in the P site and ensue the
release of the nascent polypeptide.
1. There are two classes of release factors: Class1 and
Class II RFs

2. Class I release factors recognize the stop codons and

trigger hydrolysis of the peptide chain from the tRNA in
the P site.

3.Prokaryotes have two class I release factors called RF1

and RF2.
RF1 recognizes the stop codon UAG, and RF2 recognizes
the stop codon UGA.

4.The third stop codon, UAA, is recognized by both RF1

and RF2.

5. A single class I RF called eRF1 recognizes all three

stop codons in eukaryotes.
Class II release factors(RFs) are GTPases that stimulate the
dissociation of the class I factors from the ribosome after
release of the polypeptide chain.

One class II factor called RF3 is present in prokaryotes

One class II factor called eRF3 is present in eukaryotes

Like EF-G, EF-Tu, and other translation factors , class II release

factors, RF3 or eRF3 are regulated by GTP.

The RF3 or eRF3 stimulate termination reaction in a GTP

dependent manner.
How RFs recognize stop codons? The class1 RFs has two
functions: a) recognition of the stop codon, and b)
release/hydrolysis of the nascent peptidyl chain.

A sequence of three amino acids (SPF i.e serine-proline and

phenylalanine) in RF protein recognizes the stop codon and
thus serves as a peptide anticodon.

Other function (release of the nascent polypeptide) evidently

requires a conserved GGQ (glycine-glycine-glutamine)
sequence in RF. The GGQ amino acid motif, involved in
polypeptide hydrolysis, is located adjacent to the 3' end of
the P site tRNA.

These two regions are close to each other in the absence of a

ribosome but in the presence of a ribosome, the release
factor undergoes a conformational change that would space
these two regions appropriately to serve the two functions
 Model of a type I
release factor bound to
the A site of the
ribosome. This model
illustrates the location
of a class I release
factor bound to the
ribosome.

The P site and E site

tRNAs are shown as L-
shaped surfaces.
The GGQ amino acid motif that is involved in polypeptide
hydrolysis is located adjacent to the 3' end of the P site tRNA.
The SPF peptide anticodon is located adjacent to the
anticodon loop of the P site tRNA in a position that would allow
easy access to the stop codon.
 Termination.
Release factors (RFs) recognize the stop codons and
trigger polypeptide hydrolysis from the tRNA in the P
site.

RFs: Class1(Prokaryotes:RF1 and RF2 and Eukaryotes:

RF1). Mimic tRNA

RF1 recognizes UAG, RF2 recognizes UGA. The third

stop codon UAA is recognized by both RF1 and RF2.

Class 2 factor is RF3 (prokaryptes) or eRF3 (Eukaryotes).

Putative peptide anticodon, SPF: (Ser-Pro-Phe), A

second conserved region in class1 factors is GGQ (gly-
gly-gln) motif that is required for peptide hydrolysis.
Probably it induces a change in the peptidyl transferase
center to peptide hydrolysis

RF3 function is to release RF1 from ribosome. It requires

the exchange of GDP bound to RF1 for GTP.
 Ribosome Recycling Factor, RRF and
Ribosome Recycling
After the release of the nascent polypeptide
and release factors, ribosome is still bound
to the mRNA and is left with two deacylated
tRNAs in the Psite and E site.

Ribosome Recycling: Removal of t RNA and

mRNA from ribosomes and the dissociation
of dissociation of ribosome into subunits.

RRF: Ribosome release factor then enters

the A site and and recruits EF.G GTP and IF3
may also participate in this event to
evacuate the tRNAs and dissociate the
ribosome.
Ribosomes: Molecular Machine Importance of Ribosomal RNA

Small subunit-decoding center; large subunit-catalytic center

Ribosomal RNA serves not only as structural component but also

participates in key functions of the ribosome

RNA is in the centre and proteins are at the periphery of the ribosome.
Proteins stabilize the tightly packed rRNAs by shielding the negative charges
of their sugar-phosphate backbone.

Peptidyl transferase Center is composed of entirely tRNA to suggest that peptide

bond formation is formed by RNA than by protein enzyme. The anticodon
loops of charged tRNAs and the codons of mRNA contact the 16S ribosomal
RNA but not the proteins of the small subunit

Infact two specific nts of 16S rRNA modulates the movement of mRNA-tRNA
and checks the accuracy of the codon-anticodon interaction

It is likely the contemporary ribosome may have evolved from a primitive

protein synthesizing machine that is composed of RNA only
Importance of 16S and 23S rRNA

Anticodons loops of the charged tRNAs and the

codons of the mRNA contact 16S rRNA, not the
ribosomal proteins.

16S rRNA modulates the movement of mRNA-

tRNA and the ribosome constantly and changes
its shape.

16S rRNA contains Shine Dalgarno sequence:

identifies start codon

23S rRNA catalyzes the peptide bond formation

 Regulatory Regions in Eukaryotic mRNA

The m7 GpppN cap structure at the 5 end of the mRNA, and the poly A tail at the 3
end are canonical motifs that strongly promote translation initiation.

Secondary structures, such as hairpins or pesudoknots (RNA tertiary structure

that is formed when the single stranded loop in a hair pin structure base pairs
with a complementary sequence outside of the hairpin) block translation.

Internal ribosome entry sequences (IRES) are structures that are located in the
5end of some RNAs of cellular or viral origin. Mediate cap-independent
translation.

Upstream open reading frames (uORFs) are small ORFs of some mRNAs that
normally function as negative regulators of translation from the main ORF.

Green ovals symbolize binding sites for proteins/ and or RNA regulators, which
usually inhibit but occasionally promote translation