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TRANSLATION
1. Template – m RNA
2. Substrates – 20 amino acids.
3. Source of energy – ATP, GTP, energy-
rich bond in amino acyl-tRNAs.
4. Enzymes – specific amino acyl tRNA
synthetases,
peptidyl transferase (ribozyme -28S rRNA
of 60S subunit), protein translation
factors.
5. Product – protein (polypeptide chain).
6. Localization in the cell – cytoplasm
(ribosomes).
Activation of amino acids
It requires:
1. 20 amino acids;
2. Enzymes –amino acyl tRNA
synthetases;
3. tRNAs;
4. АTP;
5. Mg²+.
Charging of tRNA
Role of tRNA
in protein
biosynthesis:
1. Transport of
amino acid to
the ribosome;
2. Adaptor
function
1. Contains dihydrouracil
residue
2. Acts as the recognition site
for specific aminoacyl tRNA
synthetase
Involved in the
binding of amino acyl
tRNA to ribosomal
surface
AMP
AMP tRNA tRNA AMP+
An amino acid An amino acid–AMP
AminoacyltRNA Synthetas
An amino acid–AMP Amino acid–tRNA
ATP Mg2+
Amino acyl–tRNA
Anticodon
3’-end
 О
Translation
Initiation
It requires the following components:
1. mRNA;
2. initiator аа-t-RNA (Met-tRNA);
3. start codon in mRNA (AUG);
4. 40S and 60S subunits;
5. GTP;
6. Eukariotic initiation factors (elF);
7. Mg²+.
Formation of 43S pre initiation complex
• 1GTP binds with eIF-2 and forms
binary complex
• Binary complex binds with met
tRNA to form ternary complex
• which binds to 40S subunit of
ribosome to form 43 S complex
• Formation of 48S initiation complex
• Binding of 43s complex to mRNA forms
48S complex
Factors are required for this:
- 5’ cap and cap binding complex
(eIF-4 recognizes cap and helps
it to attach 43 S complex)
- eIF-4 controles rate of translation
- 43 complex has helicase activity
and unwinds hairpin loops in
mRNA (using ATP)
-It also scan mRNA until not find
AUG codon
-ternary complex binds with AUG
• Formation of 80S initiation complex:
(48 S+60 S= 80S)
• Involves GTP hydrolysis:
- eIF5 – hydrolysis of GTP bound
to eIF-2 facilitate 60S association
- Initiation factors are released
Translation
Initiation
40 S
ГТФ
elF2
60 S
 О
Translation
Elongation
It requires the following components:
1. initiate complex;
2. complete set of аа-t-РНК;
3. peptidyl transferase;
4. GTP;
5. eukaryotic elongation factors (eEF);
6. Mg²+.
The protein is synthesised from N terminus to its
terminus
TRANSLATION
Elongation
Includes 3 steps:
1.binding of an aminoacyl-tRNA to the
A site ;
2.formation of a peptide bond (no ATP
is required);
3. translocation - the whole ribosome
moves one codon along the mRNA.
1.
• Met-tRNA is bound to the P site of ribosome
• mRNA codon in A site determines which
aatRNA will bind to this site
• The incoming aatRNA first combines with
eucariotic elongation factor (eEF) 1,
containing bond with GTP
• When aatRNA-eEF1-GTP complex binds to
A site GTP is hydrolyses to GDP+Pi
• eEF 1 is released
2. Amino acid on the tRNA in the A site
forms
peptide bond with Met on tRNA in the P
site
Peptidyltranferase catalyses formation of
the peptide bond using high energy
bond of aatRNA.
tRNA in A site contains growing
polypeptide chain
3. Translocation involves eEF 2-
GTP complex which binds with
ribosome casing a conformation
cnange and moves ribosome
along mRNA (on one codon)
The uncharged tRNA is released
from ribosome, growing peptidyl-
tRNA moves into P site and next
codon occupies A site of ribosome
Elongation
О
ГТФ
eEF1α
О
Peptidyl transferase
(28S р-RNA)
Н
mRNA
О
ГТФ
eEF2
5’3’
Н
ГТФ
eEF1α
ОО
ГТФ
eEF2
Н
ГТФ
eEF1α
ОН
ГТФ
eEF2eEF1α
ГТФ
ОН
ГТФ
eEF2eEF1α
ГТФ
ОН
ГТФ
eEF2
mmRNA
• Tree elongation steps are
repeated untill termination codon
moves into A site on the ribosome
TRANSLATION
Termination
It requires the following components:
1. GTP;
2. termination or stop codons – UAG,
UGA, UAA
3. Enzymes – peptidyl transferase,
translocase;
4. Eukaryotic termination factors (eRF)
Termination
5’3’
О
eRF
ГТФ
Peptidyl transferase
(28S р-RNA)
Н2О
Н
Translation
product
-ОН
Posttranslational
modifications
Many proteins are synthesized in
the inactive form (as (pre)pro-
proteins) and undergo
postsynthetic modification:
– partial proteolysis (removal of
N-terminal Met and the signal
peptide, the formation of active
forms of hormones and enzymes);
– combining the protomers and
the formation of the quaternary
structure of proteins;
– the formation of intra- and inter-chain
S-S bonds;
– covalent attachment of cofactors to
the enzymes;
– glycosylation (hormones, receptors,
others);
– the modification of amino acid
residues:
hydroxylation of Pro and Lys
(collagen);
iodination (thyroid hormones);
carboxylation (blood clotting factors);
Corynebacterium
diphtheriae
Tabulettae
Examples of inhibition of template biosynthesizes
Doxorubicin - it is binding to DNA, generating
free radicals, thereby changing the structure of
DNA in mammalian cells.
Mitomycin С – binds tightly with both strands of
DNA
Inhibitors of replication
Inhibitors of trancription
Rifampin inhibits the initiation of transcription by
binding to prokaryotic RNA polymerase.
Rifampin is useful in the treatment of tuberculosis.
Dactinomycin (known to biochemists as actinomycin
D) binds to the DNA template and interferes with the
movement of RNA polymerase along the DNA.
ANTIBIOTIC TARGET EFFECT
Erythromycin Prokaryotic 50S
subunit
Blocks translocation
Chloramphenicol Prokaryotic, peptidyl
transferase
Blocks peptide bond
formation
Streptomycin Prokaryotic ribosome Inhibits initiation
Tetracycline Prokaryotic ribosome Blocks binding
aminoacyl tRNA with
A-site
Antibiotics can be used to probe protein synthesis at various stages.
Inhibitors of translation
Diphtheria toxin of Corynebacterium diphtheriae catalyzes
the ADP-ribosylation of EF-2 and this modification inactivates
EF-2 in mammalian systems.
Ricin (toxin from the castor bean) inactivates eukaryotic 28S
ribosomal RNA.
Alpha-amanitin ( from Amanita phalloides) inhinits RNA pol
II
Toxins

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Translation DNA

  • 2. 1. Template – m RNA 2. Substrates – 20 amino acids. 3. Source of energy – ATP, GTP, energy- rich bond in amino acyl-tRNAs. 4. Enzymes – specific amino acyl tRNA synthetases, peptidyl transferase (ribozyme -28S rRNA of 60S subunit), protein translation factors. 5. Product – protein (polypeptide chain). 6. Localization in the cell – cytoplasm (ribosomes).
  • 3. Activation of amino acids It requires: 1. 20 amino acids; 2. Enzymes –amino acyl tRNA synthetases; 3. tRNAs; 4. АTP; 5. Mg²+. Charging of tRNA
  • 4. Role of tRNA in protein biosynthesis: 1. Transport of amino acid to the ribosome; 2. Adaptor function
  • 5. 1. Contains dihydrouracil residue 2. Acts as the recognition site for specific aminoacyl tRNA synthetase Involved in the binding of amino acyl tRNA to ribosomal surface
  • 6. AMP AMP tRNA tRNA AMP+ An amino acid An amino acid–AMP AminoacyltRNA Synthetas An amino acid–AMP Amino acid–tRNA ATP Mg2+
  • 8. Translation Initiation It requires the following components: 1. mRNA; 2. initiator аа-t-RNA (Met-tRNA); 3. start codon in mRNA (AUG); 4. 40S and 60S subunits; 5. GTP; 6. Eukariotic initiation factors (elF); 7. Mg²+.
  • 9. Formation of 43S pre initiation complex • 1GTP binds with eIF-2 and forms binary complex • Binary complex binds with met tRNA to form ternary complex • which binds to 40S subunit of ribosome to form 43 S complex
  • 10. • Formation of 48S initiation complex • Binding of 43s complex to mRNA forms 48S complex Factors are required for this: - 5’ cap and cap binding complex (eIF-4 recognizes cap and helps it to attach 43 S complex) - eIF-4 controles rate of translation
  • 11. - 43 complex has helicase activity and unwinds hairpin loops in mRNA (using ATP) -It also scan mRNA until not find AUG codon -ternary complex binds with AUG
  • 12. • Formation of 80S initiation complex: (48 S+60 S= 80S) • Involves GTP hydrolysis: - eIF5 – hydrolysis of GTP bound to eIF-2 facilitate 60S association - Initiation factors are released
  • 14. Translation Elongation It requires the following components: 1. initiate complex; 2. complete set of аа-t-РНК; 3. peptidyl transferase; 4. GTP; 5. eukaryotic elongation factors (eEF); 6. Mg²+. The protein is synthesised from N terminus to its terminus
  • 15. TRANSLATION Elongation Includes 3 steps: 1.binding of an aminoacyl-tRNA to the A site ; 2.formation of a peptide bond (no ATP is required); 3. translocation - the whole ribosome moves one codon along the mRNA.
  • 16. 1. • Met-tRNA is bound to the P site of ribosome • mRNA codon in A site determines which aatRNA will bind to this site • The incoming aatRNA first combines with eucariotic elongation factor (eEF) 1, containing bond with GTP • When aatRNA-eEF1-GTP complex binds to A site GTP is hydrolyses to GDP+Pi • eEF 1 is released
  • 17. 2. Amino acid on the tRNA in the A site forms peptide bond with Met on tRNA in the P site Peptidyltranferase catalyses formation of the peptide bond using high energy bond of aatRNA. tRNA in A site contains growing polypeptide chain
  • 18. 3. Translocation involves eEF 2- GTP complex which binds with ribosome casing a conformation cnange and moves ribosome along mRNA (on one codon) The uncharged tRNA is released from ribosome, growing peptidyl- tRNA moves into P site and next codon occupies A site of ribosome
  • 21. • Tree elongation steps are repeated untill termination codon moves into A site on the ribosome
  • 22. TRANSLATION Termination It requires the following components: 1. GTP; 2. termination or stop codons – UAG, UGA, UAA 3. Enzymes – peptidyl transferase, translocase; 4. Eukaryotic termination factors (eRF)
  • 26. Many proteins are synthesized in the inactive form (as (pre)pro- proteins) and undergo postsynthetic modification: – partial proteolysis (removal of N-terminal Met and the signal peptide, the formation of active forms of hormones and enzymes); – combining the protomers and the formation of the quaternary structure of proteins;
  • 27. – the formation of intra- and inter-chain S-S bonds; – covalent attachment of cofactors to the enzymes; – glycosylation (hormones, receptors, others); – the modification of amino acid residues: hydroxylation of Pro and Lys (collagen); iodination (thyroid hormones); carboxylation (blood clotting factors);
  • 29. Doxorubicin - it is binding to DNA, generating free radicals, thereby changing the structure of DNA in mammalian cells. Mitomycin С – binds tightly with both strands of DNA Inhibitors of replication
  • 30. Inhibitors of trancription Rifampin inhibits the initiation of transcription by binding to prokaryotic RNA polymerase. Rifampin is useful in the treatment of tuberculosis. Dactinomycin (known to biochemists as actinomycin D) binds to the DNA template and interferes with the movement of RNA polymerase along the DNA.
  • 31. ANTIBIOTIC TARGET EFFECT Erythromycin Prokaryotic 50S subunit Blocks translocation Chloramphenicol Prokaryotic, peptidyl transferase Blocks peptide bond formation Streptomycin Prokaryotic ribosome Inhibits initiation Tetracycline Prokaryotic ribosome Blocks binding aminoacyl tRNA with A-site Antibiotics can be used to probe protein synthesis at various stages. Inhibitors of translation
  • 32. Diphtheria toxin of Corynebacterium diphtheriae catalyzes the ADP-ribosylation of EF-2 and this modification inactivates EF-2 in mammalian systems. Ricin (toxin from the castor bean) inactivates eukaryotic 28S ribosomal RNA. Alpha-amanitin ( from Amanita phalloides) inhinits RNA pol II Toxins