1

TRANSLATION
The central dogma of life

DNA

transcription

mRNA

translation

protein

THE GENE

The code has 4 letters- A,C,G & T (4 bases)

The code is composed of triplet codes/codons. eg - AGC, CTC, TGG
Each codon code for one amino acid.
3
There are 64 diferent codones. ( 4 =64 )

But only 61 of them code for amino acids.

The other 3 do not- stop codons
A series of codons in DNA that coding for a protein is a GENE.

Characteristics of the genetic code

Unambiguous (specific)
The code occur as codons. Each codon code for only one amino acid.
Degenerate
But a amino acid may have more than one codon. eg;- Ala - 4 codons
Non overlapping
Code is read,
1. From a fixed starting point. (AUG)
2. 3 bases at a time
3. No punctuation (continuously)
Universal (almost)
The code is almost the same for all the living organisms.

Why almost?
Because there are minor variations
The amino acid for start codon (AUG)
In eukaryots- Meth
In prokariotes- fMeth
Variations in mitochondrial genome, chloroplast & some ciliated protozoa. Eg;AGA is an stop codon in mitochondrial genome
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Translation
Converting the coded message in the mRNA ,the amino acid sequence of the protein
Translation requires-mRNA, tRNA, ribosomes, initiation factors(proteins),enzymes
AA ,energy

tRNA
3 end carries the amino acid.
base pairing is antiparallel.(3rd base of codon
pair with 1st base of anticodon)
wobble hypothesis

pairing of 3rd base in codon with 1st base of anticodon

not strictly by Watson-Crick rule.(loosely bound to anticodon)
3rd base of codon is called Wobble Base

Therefore more than one tRNA can bring

one amino acid
eg;- anticodon- (3) XYU (5)
codon
-(3) YXA (5)
(3) YXG (5)
For one amino acid

Formation of amino acyl tRNA

Step 1 :- Activation of AA

( AA+ ATP Aminoacyl AMP+ PPi)

Step 2 :- Transfer of aminoacyl group to tRNA

( AminoacylAMP+ tRNA AminoacyltRNA+ AMP)
Both activities are catalized by Aminoacyl tRNA synthetase
It has a proof reading activity to ensure that the correct amino acid is bound to
the tRNA.
1st check (represents the 1st step)
If wrong amino acyl AMP is bound to the enzyme,
it is hydrolyzed to AA and AMP

2nd check (represents the 2nd step)

If wrong amino acid is bound to tRNA,
The ester linkage of amino acyl tRNA is hydrolyzed.

There are minimum of 20 aminoacyl tRNA synthetases, each specific for one
amino acid.
But there are 60 different tRNAs. The tRNAs that can bind the
same amino acid are recognized by the same enzyme.
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Specific nucleotides on tRNAs are involved in this recognition by the enzyme.this
is called second genetic cord

Aminoacyl tRNA
Ribosomes :- eukaryotic-

80 S 40 S+60 S

prokaryotic-

70 S 30 S +50 S

amino acid

Ester bond

Prokaryotic DNA translation.

Three steps :-

initiation elongation termination

Initiation
1.

Ribosomes separate(IF1 & IF3 help and prevent recombination)

70 S 30 S +50 S

2. 30S subunit join mRNA near 5 end, the P site opposite the start/initiation
codon(AUG). (Shine- Delgano sequence just upstream of AUG help in oppositioning)
3. fMet-tRNA bind to AUG forming 30S initiation complex. ( GTP bound IF2 help this)
4. 50S subunit bind to 30S initiation complex, GTP hydrolyzed, 70S initiation complex
formed.

1
A 2
5 U
P
U3A
X
A
C0

3
X
G X X
S

30S initiation complex

1
5
0
U
A
S32 3
PA
A
U0X X
C
GSX X

70S initiation complex

12

AG
23
5U
A
A
UA
XX
P
A
C
GXX

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Elongation
1. Elongation factor Tu(EF Tu), GTP & aminoacyl-tRNA, form a complex and and bind to
ribosome at A site.
2. If anticodon does not match, complex dissociate.
3. If matches, GTP is hydrolyzed EF-Tu-GDP complex leave the ribosome. Aminoacyl
tRNA remains at A site
12
4. Peptide bond formation
U
50S subunit of ribosome has peptidyl-transferase activity.
AG
23
A
A
Ester bond between fMet & tRNA is broken , fMet is
UXX
C
transferred to A site, peptide bond is formed between 1 st &
GA
XX
nd
2 amino acid at A site.
5. Translocation of ribosome
GTP binding EF-G (a translocase) hydrolyse GTP and provide
energy.
Ribosome changes its shape and moves to the next triplet
1 23
cord, in 3 direction.
Dipeptide now occupy P site, tRNA at AUG codon leaves the U
A A G
3
2 C
ribosome.
A
G
C
6. New amino acyl tRNA arrives at empty A site.
UX X
A X
7. The process continues till the stop codon is reached.
GXA

5 PA 3

3
PA

Termination

1. When A site occupy a termination codon(UAA,UGA,UAG) a Releas Factor(RF) binds to

it(prokaryotes- RF1, RF2,RF3 , eukaryotes- eRF)
2. Protein-tRNA bond hydrolyzed releasing the protein & tRNA.
3. Ribosome dissociate to start a new cycle.

6
45 718
3

R
U 3
8F
5 7G
A
XA
XPA
A

6
45 78
3

R
G
F
5A 7 8 U
XA
A X PA
XXG

XXG

Polyribosome/ Polysome= several ribosomes attached to one mRNA.

Can be free in cytosol or attached to ER (RER)

# in eukaryotes initiator tRNA carries a Met that is not formylated.

# In both prokaryotes & eukaryotes fMet & Met are removed before protein is completed.

Antibiotics that inhibit ribosomal protein synthesis

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Drug

subunit

Target organism

effect

Streptomycin

30S

prokaryotes

Interfere with binding of fMet-tRNA toribosome

,thus prevent correct initiation

Tetracycline
Chloramphenicol
Erythromycin
Puromycin

30S
50S
50S
50S

prokaryotes
prokaryotes
prokaryotes
prokaryotes &

Stop aminoacyl tRNA binding

Inhibit peptidyl transferase
Inhibit translocation
Structural analogue of tyrosinyl-tRNA

Cycloheximide

60S
60S

eukaryotes
eukaryotes

incorporate at c-treminal & stop elongation

Inhibit peptidyl transferase

Effect of diphtheria toxin on protein synthesis

Organism- Corynebacterium diphtheria, a bacterium
The toxin bind to receptors on mucosal cell surface & is proteolytically cleaved.
one fragment the cell and
inactivate elongation factor 2 (EF2)
It is equivalent to prokaryotic EF-G
EF2 is required for the GTP driven translocation of ribosome along mRNA.
Stop protein synthesis & kills the cell.
Act as an enzyme & small amount is sufficient for a great damage.
(any 6 x 5= 30)
Signal sequence
a. 20-25 AAs long portion at the amino terminal end of export proteins.
b. Contain high proportion of hydrophobic AAs
c. helps export proteins from inside the cell to the out side.

Post translational modifications

Changes that a translated protein undergo to form the final Biologically Active Protein
1. Amino/carboxyl terminal modifications
a. Formyl group of amino terminal Met frequently removed.
b. Acetylation of amino terminal residue in many eukaryotic proteins.
2. Removal of Signal sequence. Eg:- ALA synthase , insulin, collagen
3. Formation of disulfide bonds between Cys: residues Eg Insulin ,collagen
4. Proteolytic cleavage of parts of polypeptide chain.
a. Removal of the c-peptide from insulin.
b. Removal of the hexapeptide from the amino terminal converts trypsinogen to
active trypsin.
c. Removal of the 44 AAs from the amino terminal converts pepsinogen to active
pepsin.
5. Covalent adition of prosthetic groups.
a. addition of biotin to Acetyl CoA Carboxylase
b. addition of heme group to Cytochrome C
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6. Modifications of individual AAs
a. Phosphorylation of OH groups of Ser, Thr & Tyr
I.
Milk protein casein has many Phosphoserines.
Net negativity of the protein increases.
Help bind Ca2+
II.
Uncontrolled Phosphorylation of certain Tyr residues of some proteins
induce cancer.
Eg:- erbB oncogene product- erbB protein (a membrane receptor)
continuously phosphorylate proteins & lead to adenocarcinomas
b. Hydroxylation
Proline & Lysine residues of procollagen are hydroxylated (by Prolyl Hydroxylase,
Lysyl Hydroxylase) during the maturation process.
This process requires Ascorbic acid.
A deficiency leads to poor hydroxylation of collagen.
Poor cross linking between fibrils leads to Scurvy

7. Glycosylation
a. enzymetic glycosylation at
Amide nitrogen of Asparagine
Hydroxyl groups of Ser or Thr
b. nonenzymetic Glycosylation of Hb
occur at N-terminal NH2 groups.
Glycosylated Hb concentration reflects glycaemic status during entire life
span of a RBC .(accurately monitor 6th to 8th week of time)
Useful to check glycaemic control of diabetis.

8. I- cell disease
A carbohydrate moiety- an oligosaccharide containing mannose 6-PO4 is
attached to the lysosomal proteins.
mannose 6-PO4 is bound by a specific Glycosyltransferase.

mannose 6-PO4 acts as a Targeting signal that is identified by receptor that

target the protein into lysosomes.
Lack of the Glycosyltransferase lead to glycoproteins without the
mannose 6-PO4 residue.
Targeting process of enzymes to lysosome is deficient.
The enzymes are secreted out of the cell.
Lysosomes accumulate partly digested materials that manifest as
Inclusion bodies.
Repeat campaign
A/L
2010
Any 6
x 5=30

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MCQ

1.

T/F
a) Proteins are synthesized from amino to
carboxyl end.
b) Streptomycin inhibits protein synthesis by
binding to m- RNA.
c) ) The catalytic activity of the larger
ribosome subunit is
responsible for the elongation of the new
polypeptide
chain.
d) A single AA may bind more than one type of
tRNA.
e) Methionine is coded by a single codon.
a) T
b) F (30 s ribosomal subunit)
c) T -50s subunit has peptydyl transferase
activity
d) T
e) T (Met & Trp has single codons)
f) Met. is retained in most eukaryotic proteins.
g) Splicing of exon is an example of a post
translational
modification.
h) Disulphide bonds are formed in insulin in
post transational
modification.
i) Secretory proteins initially contain a signal
sequence.
j) Insulin undergoes post translational
modifications.
f) F Met & fMet are removed before protein is
completed
g) F post transcriptional
h) T
i) T
j) T
2. Regarding t RNA
a) It undergoes post translational
modifications.
b) It shows dual specificity.
c) Single tRNA molecule recognizes more than
one AA.
d) tRNA travels along the mRNA in
transcription.
e) Translation is stopped when it binds to a
stop codon.
a) F
b) T-can translate language of nucleic acid to
proteins
c) F
d) F -( P site to A site then E site of ribosome
small subunit)
e)F when it binds to release factor.
3. Eukaryotic translation requires
a) Formylated methionine.

b) GTP.
c) ATP.
d) Acyl tRNA synthase.
e) Sigma factor.
a) F - Met
b) T
c) T
d) T
e) F for prokaryotes
4. T/F
a) 70s ribosomes are essential for eukaryotic
protein
synthesis.
b) Eukaryotic genes are split genes.
c) A triple code cods for more than one amino
acid.
d) A gene on a DNA codes for a protein.
e) Protein is synthesized in 5-3 direction.
a) F
b) F
c) F
d) T
e) T
5. T/F
a) Inhibitors that bind 80s ribosomes can be
used as
treatment for the bacterial infections.
b) Erythromycin inhibits the translocation.
c) Penicilline inhibits the protein synthesis in
prokaryotes.
d) Diphtheria inhibits the translocation in
eukaryotes.
e) Streptomycin inhibits the initiation of
translation.
a) F
b) T
c) F
d) T It inactivates EF2
e) T-inhibits binding of fMet tRNA
6. T/F
a) Different proteins are synthesized by exon
shuffling.
b) On eukaryotes both cytoplasmic and
mitochondrial
ribosomes are 70s.
c) Eukaryotic small ribosomal subunit binds mRNA by base
pairing.
d) Lac operon consists of structural genes and
regulatory
genes.
e)Eukaryotic 40s ribosome subunit binds to 5
cap region of mRNA
a) T (Causes formation of new exons and thereby
new proteins)

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b) F
c) F
d) T
e) T
7. Regarding translation
a) Amaino acyl tRNA synthase has a proof
reading activity.
b) Shine-Dalgarno sequence base pair with 3
end of rRNA of the 40s subunit
c) Eukaryotic translation begins with formyl
methionine.
d) Tetracyclin is an inhibiter of prokaryotic
translation.
e) Aminitin is an eukaryotic protein synthesis inhibiting
compound.
a) T
b) F it base pair with 30s subunit of
prokaryotes
c) F Met
d) T
e) T
8. Regarding the genetic code,
a) Single codons code for more than one
amino acid.
b) The codons for methionine & formyl
methionine are
different.
c) The mitochondrial DNA codes for electron
transport
proteins.
d) 61 codons constitute the genetic code
e) Degeneracy is mostly in1st base
a) F
b) F
c) T
d) F- 64
e) F 3rd base
9. T/F
a) Vit K is required for post-translational
modifications of clotting factors.
b) Defective post translational modification of
procollagen
can lead to delayed wound healing.
c) Removal of signal sequence of secretory
proteins is a post
translational modification.
d) Biologically active form of insulin contains
the C peptide
sequence.
e) The signal sequence is present in histones
a) T
b) T
c) T
d) F C peptide is removed
e) F

SEQ
Q. . Explain the role of aminoacyl tRNA

synthetases in protein synthesis.(50)

(2003 main)

The process of linking the appropriate

tRNA to its specific AA is considered
the 2nd genetic code.

It mans that AAs are activated by

likage to proper tRNA carrier before
translation.

This t-RNA charging process occurs

in cytosol.

It is also called AA acylation process.

This is catalized by amynoacyl t-RNA

synthetase using ATP.

There are specific enzymes for each

AA & corresponding t-RNA.

If one AA can bind to many tRNA ,

same enzyme recognizes all of those
tRNA.

So there are minimum of 20 aminoacyl

tRNA synthetases.

Charging of t-RNA occurs in 2 steps.

1-Activaton of AA

2-Transfer of Aminoacyl group to tRNA

There are AA ,ATP & t-RNA binding sits

in the enzyme.

1st AA & ATP bind to their sits in the

enzyme.

ATP looses two phosphate groups &

binds to AA as AMP to form aminoacylAMP

This is called activation of AA

Then uncharged t-RNA binds to its site

in enzyme

Aminoacyl group is now transferred to

the t-RNA

& release AMP, to form aminoacyl tRNA

This enzyme has proofreading ability.

When a wrong AA is attached ,the

mistake can be corrected at both
stages.
Q. Explain the role of tRNA in the
accurate reading of the nucleotide.(40)
(AL1999 CAT2)

t-RNA has dual specificity.

It carries AA to site of protein synthesis

by forming aminoacyl t-RNA

t-RNA has large number of modified

bases ;

bcz of the unusual bases, it folds into

3dimentional shape ,which forms an
AA binding site.

t-RNA can recognize the codon in mRNA.

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Bcz it has modified, unusual bases it

folds into 3dimentional shape, which
forms an anticodon site.

Q .Explain the biochemical basis for

the action of erythromycin as an
antibacterial agent.(50)
(1998
CAT 2)

Erythromycin interfere with elongation

process of prokaryotic translation
process.thus inhibits protein syn.
70s ribosomes which have 30s & 50s
subunits are involved in prokaryotic
protein syn.
50s subunit acts as Peptydyl
Transferase which catalyzes
breakdown of ester bond between AA
& t-RNA, which are on P site.
Then AA is transferred to A site
& forms a peptide bond with the AA in
there.
Ribosome moves to next codon in 3
direction on m-RNA.It is called
translocation.
Now A site is empty & can bind a new
amynoacyl t-RNA
Erythromycin binds with 50s subunit &
inhibits translocation.
From this point m-RNA is not read &
protein syn. Is stopped.

Q ..List the steps involving in the

synthesis of proteins in the rough ER.
(60) (2000 CAT 2)
(RER means they are asking about eukaryotic
protein syn.)

Eukaryotic protein syn. Occurs in 3

steps.

Initiation ,Elongation ,& Termination.

Many protein factors required (a large

number than prokaryotes)

Eg- eIF , eEF ,eRF

Initiation -40s & 60s subunits
separate.IF s help this & prevent
recombining.
40s subunit complex binds to 5 cap
region of mRNA
Moves along mRNA in 3 direction
looking for an AUG start codon.
It is called Scanning for initiation
codon.
P site aligns opposite AUG.
Met t-RNA binds to P site & then 60s
subunit comes & binds to 40s subunit
to form complex
Elongation EF ,GTP & aminoacyl tRNA form acomplex.

At the A site, if the anticodon does not

match , complex dissociates.

If it match properly, GTP hydrolysis

occurs.

EF & GDP complex leave the ribosome

Formation of peptide linkage between
AAs

The ester bond between Met & t-RNA

is broken

& Met transferred to A site by peptydyl

transferase activity.

Translocation of ribosome

Ribosome travels by the distance of

one codon in the 3 direction with the
help of EF2 & GTP.

Now the dipeptide occupies P site & A

site is empty. & tRNA at AUG codon
leaves the ribosom

A new aminoacyl tRNA now arrive

empty A site

Teheprocess continues till the stop

codon is reached.
termination when A site occupies a
termination codon (UAG , UAA , UGA)

releasing factor (eRF) binds A site.

Termination occurs.

Protein- tRNA bond hydrolyzed.

& release protein & t-RNA.

POST TRANSLATIONAL MODIFICATIONS

Q . List 3 post translational

modifications and explain their
importance (60)
1. N/C terminal modification
2. Removal of signal sequence
3. Formation of disulphide bonds (b/w
cystein residues)
4. Proteolytic cleavage of parts of
polypeptide chain
5. Covalent addition of prosthetic group
6. Glycosylation/attachment of
carbohydrates
7. Modification of individual A.A
(phosphorylation)
8. Hydroxylation of A.A
1) Removal of the signal sequence
Export proteins are modified by
the presence of signal sequence
Signal sequence targets the
protein into RER
After the protein enter RER, signal
sequence no longer needed
It is removed producing the
functional protein
2) Formation of disulphide bond (b/w
cystein residues)
Insulin is synthesized as a single
chain precursor prohormone

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It folds allowing the disulphide

bonds to form
This is one of the steps where it
converted to biologically active
form e.g. insulin

3) Proteolytic cleavage of polypeptide

chain
E.g. removal of the C-peptide from
insulin-or Removal of the hexapeptide from
the amino terminal, converts
trypsinogen to active trypsin -or Removal of N and C terminal
propeptides in procollagen
Important for aligning in collagen
synthesis
Proteolytic cleavage forms the
biological active form
regarding proteases it should occur
in their proper site of action
4) Covalent modification of prosthetic
groups
E.g. addition of biotin to Acetyl CoA
carboxylase
Addition of heme group to cyt.C
Required for their biological
activity
5) Glycosilation
a)
Eg. -: enzymatic glycosilation at
amide nitrogen of asparagines /
hydroxyl group of Ser or Thr or Non enzymatic glycosilation of Hb
GlycosilatedHb concentration
reflects glycaemic status during
entire life span of a RBC
Useful to check glycaemic control
of diabetes
b)
The CHO moiety helps to target
certain enzyme proteins into
lysosomes
Oligosaccharides containing
mannose-6-PO43- which act as a
targeting signal identified by
receptor that target the protein
into lysosomes
Lack of particular
glycosyltransferase leads to
glycoprotein without the
mannose-6-PO43-
Targeting process of enzymes to
lysosomes is deficient

The enzymes are secreted out of

cells
Lysosome accumulate partly
digested material that manifests
as inclusion bodies

6) Phophorylation of OH- groups

Add negative charges to the
proteins serving various functions
Eg. Milk protein casein has many
phophoserines
Help bind Ca2+
7) Hydroxylation
E.g. proline and lysine residues in
procollagenhydroxylated during
maturation process
This process requires ascorbic acid
(Vit C)
A deficiency leads to poor
hydroxylation of collagen
Poor cross linking b/w fibrils
Leads to scurvy
Q. Give post translational
modifications occur in conversion of
prepro insulin to its biologically
active form. ( 50 )
Gene cording for insulin is
transcribed to mRNA in nucleus.
After moving into cytoplasm,
translation is initiated on
cytoplasmic ribosomes,
with formation of N terminal
hydrophobic signal sequence that
directs mRNA & the ribosome to
RER.
Signal peptide penetrates the RER
membrane.
Further elongation directs the
polypeptide into RER lumen,
Resulting in formation of Prepro
insulin.
Prepro insulin is a biologically
inactive, long single polypeptide
chain containing a signal sequence
& a C peptide that bends the
chain.
Signal sequence is
cleaved/trimmed by protease
activity & Proinsulin formed in
the RER lumen.
C peptide is responsible for the
proper folding of pro insulin

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forming a short A chain & a long B

chain.
Due to the folding, two interchain
disulphide bonds formed between
sys. residues in A & B chains; &
One intrachain disulphide bond
formed in A chain.
Pro insulin is transported from RER
to golgi complex.
C peptide is removed forming
biologically active Insulin.
Insulin & cleaved C peptide is
secreted out of the cell via
secretory granules by exocytosis.

Q . Explain the role of Vit C in post

translational modifications (50) 2002
CAT 2

Vit C is required as a reducing agent

that involve in post translational
modifications during collagen syn.

It is needed to convert pre-pro

chains to biologically active collagen.

After pre-pro chains enter to RER

lumen & remove their signal
sequences , selected prolin & lysine
residues are hydroxilated
enzymatically
This is a covalent modification & it is
called Hydroxilation.
This requires Vit C.
Proline prolyl hydroxilase
Hydroxyproline
Vit C
Lysine
lysyl hydroxilase
Hydroxilysine
In the case of Vit C deficiency collagen
fibers cannot be cross-linked ,
greatly decreasing the tensile strength
of the assembled fiber.
This deficiency disease is known as
scurvy.
Dark bruises appear on the limbs as a
result of increased capillary fragility.

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