DR VISHNU KUMAR

1. Translation or protein synthesis
DR. Vishnu Kumar
PROFESSOR & HEAD,
DEPT.OF BIOCHEMISTRY
MPTMC, SIDDHARTH NAGAR
COMPETENCY NUMBER BI 7.2

2. LEARNING OBJECTIVES
After completion of this lecture learner
should be able to define/ describe:
• Components needed for protein synthesis
• Steps of protein synthesis
• Peptide bond formation
• Post translational modification
• Inhibition of protein synthesis
• Inhibition of N.A. synthesis
• Drugs used in cancer chemotherapy

3. Translation or protein synthesis
• Components needed for protein synthesis.
• 1) Amino acids.
• 2) tRNA.
• 3) mRNA.
• 4) Aminoacyl tRNA synthase.
• 5) Ribosome.
• 6) Protein factors.
• 7) ATP / GTP.

4. Translation in pro and eukaryotes
are similar with some differences
1) the 1st amino acid is Methionine in
eukaryotes but N formyl Methionine in
prokaryotes.
2) Shine- Delgarno sequence is present
at the start site in prokaryotes, Kozak
sequence in eukaryotes.
3)Eukaryotic Ribosomes are larger ( 80
S).

5. Differences
• In Prokaryotes protein synthesis begins
before transcription is complete i.e.
transcription and translation go on
simultaneously.
• In Eukaryotes transcription is nuclear one
and translation is occurs in cytoplasm. So
simultaneous transcription and translation is
not possible. Also there is necessity of
generation of mRNA from hnRNA.

6. Codon recognition by the tRNA
• Recognition of a particular codon in an
mRNA sequence is done by the anti
codon sequence of tRNA.
• Some tRNAs recognize more than one
codon for a given amino acid.
• Binding of anti codon and codon follows
the rules of complementary and anti
parallel binding.
• Codons are read in 5’- 3’ direction by an
anti codon pairing in 3’ – 5’ direction.

9. Steps of protein synthesis
• 5 steps.
• Activation of amino acid.
• Initiation.
• Elongation.
• Termination.
• Post translational modification.
• Ribosome serves as machinery for
protein synthesis

10. Activation of amino acid
• Amino acid + ATP  (amino acyl tRNA
synthatase )  E-AMP- amino acid.
• E- AMP- amino acid + tRNA (E ) 
amino acyl tRNA. Enzyme and AMP will
be released.
• This is a family of enzymes. Each
member of the family recognizes a
specific amino acid and the tRNA that
corresponds to that amino acid.

11. Initiation of protein synthesis
• At first the ribosome selects the mRNA
and binds to it. The ribosome finds the
correct reading frame on the mRNA and
translation begins. The process
involves tRNA, rRNA, mRNA , at least 9
eukaryotic initiation factors ( e .i .f s )
and also GTP, ATP, amino acids.
• Prokaryotes have 3 initiation factors.

13. A and P sites of ribosome
• The ribosomes have two binding sites
for the tRNA molecule. During
translation the A site binds with the
incoming amino acyl tRNA as directed
by the codon currently occupying this
site.
• the P site is occupied by the tRNA
containing the polypeptide chain that
has already been synthesized.

16. Sub steps of initiation
• Ribosomal dissociation.
• Formation of 43 S pre initiation
complex.
• Formation 48 s initiation complex.
• Formation of 80 s initiation complex.

19. Ribosomal dissociation and
formation of 43S pre initiation
complex
• Two initiation factors eif 3 and 1A bind
to 40S subunit. This favors dissociation
of the 80S ribosome into 40S and 60S.
• 40S ribosome now binds to GTP, eif 2
and met tRNA to form 43S pre initiation
complex.
• Met tRNA is the tRNA involved in
binding to the initiation codon AUG.

20. Formation of 48S and 80S
complex
• mRNA binds to the 43S pre initiation
complex, and along with it eif 4F, 4A
and 4B bind  48S initiation complex.
Now this complex scans the mRNA for
a suitable initiation codon.
• Binding of 60S subunit with 48S will
form 80S ribosome with release of
initiation factors and GTP.

23. Elongation
• A cyclic process, involves several
steps catalyzed by proteins called
elongation factors (e.e.f ).
• The steps are:
• Binding of amino acyl tRNA at the A
site
• Peptide bond formation.
• Translocation.

25. Binding of amino acyl tRNA to
the A site
• At the end of initiation 80 S ribosome
has A site free. Proper amino acyl tRNA
will bind at the A site by proper codon
recognition.
• EF1α and GTP allow amino acyl tRNA
to enter the A site. GTP is hydrolyzed to
GDP and Pi.

29. Peptide bond formation
• The new amino acid at the A site
attacks the carboxyl group of the amino
acid at the P site and form peptide
bond. This reaction is catalyzed by
Peptidyl- Transferase. The peptide
bond is formed at the A site and the
growing peptide chain is now at the A
site.
• Peptidyl Transferase is a Ribozyme.

32. Translocation
• Upon removal of the peptidyl moiety
from the tRNA from the P site, the tRNA
quickly dissociates from the P site
through E site.
• The newly formed peptidyl TRNA at the
A site is now translocated to the empty
P site by eef2 and GTP.
• Now the A site is free for another amino
acyl tRNA to bind to the next codon.

34. Energy required for formation of
one peptide bond
• 1) hydrolysis of 2 ATP molecules in the
formation of amino acyl tRNA.
• 2) 1 GTP for entry of amino acyl tRNA
to the A site and 1 GTP for
translocation step.
• So there is need for 4 high energy
phosphates for synthesis of one
peptide bond.

35. Termination
• After many cycles of elongation , when
protein synthesis is complete a
nonsense termination codon of mRNA (
UAA, UAG, UGA ) appears at the a site.
• Normally there is no tRNA with an anti
codon capable of recognizing such a
termination signal.
• Releasing factors (erf ) can recognize
the termination signal in the A site.

36. Termination
• The releasing factor in conjugation with
GTP and peptidyl transferase promotes
hydrolysis of the bond between the
peptide and the tRNA occupying the P
site.
• Thus a water molecule instead of an
amino acid is added.
• This releases the protein and tRNA
from the P site.
• 80S ribosome dissociates into 40S and
60S. mRNA is also released from the
ribosome.

38. Polyribosome
• Many ribosomes can translate the same
mRNA simultaneously.
• Multiple ribosome on the same mRNA
molecule form a poly ribosome or
polysome.

40. Post translational modification
• After translation the newly synthesized
protein may not be in the the active form. To
make it active some modifications are
required:
• 1) loss of signal sequence.
• 2) folding and S-S formation.
• 3) proteolytic processing.
• 4) modification of individual amino acid and
triple helix formation.
• 5) attachment of carbohydrate side chain.

41. Post translational modification
• Loss of signal sequence.
• Some proteins have 15 – 30 amino acid
residues at the beginning which direct the
protein to its proper destination.
• This signal sequences are ultimately
removed by specific peptidases.
• Example  insulin is synthesized with 23
amino acid signal peptide, which is removed.
The signal peptide is also called leader
sequence.

42. Folding and S-S bridge formation
and proteolytic processing
• Insulin is synthesized as a single chain
prohormone in the ribosome. Folding
and S-S bridge formation occurs in the
endoplasmic reticulum.
• In the Golgi apparatus a specific
protease clips out the segment that
connects two chains to form the
functional insulin molecule.

45. Triple helix formation and
modification of amino acids
• Collagen is synthesized as single chain
procollagen molecule. Three
procollagen molecules align
themselves to form the triple helix.
• Specific enzymes carry out
hydroxylation and oxidation of proline
and lysine residues to provide cross
linking and greater stability.

47. Inhibition of protein synthesis
• 1) Gentamycin, Streptomycin,
Neomycin, Tetracycline bind
irreversibly with 3oS ribosomal subunit
blocking initiation of protein synthesis.
• 2) Erythromycin, Chloramphenicol 
bind to 50S ribosomal subunit inhibit
elongation.
• 3) Puromycin  inhibits peptide bond
formation.

48. Inhibition of N.A. synthesis
• 1) Norfloxacin, O- Floxacin,
ciprofloxacin, lomefloxacin  inhibits
topoisomerase (DNA gyrase ) in
bacteria.
• 2) Nitrofurantoin, Metronidazol 
causes DNA cleavage in bacteria.
• Rifampicin, actinomycin, α aminitin
inhibit transcription.

49. Drugs used in cancer
chemotherapy
• 1) methotrxate  inhibits dihydrofolate
reductase
• 2) 5 fluorouracil, 5 iodouracil, 6
azauridine, allopurinol  inhibit
Pyrimidine synthesis.
• 3) 6 thioguanine, 6 mercaptopurine 
inhibit purine synthesis.
• Cyta arabine  inhibits replication.