Translation is the process by which cells make proteins. It involves three key factors: m RNA, t RNA and r RNA.
Elongation factors are required to add amino acids to polypeptide chain.
EF-Tu binds aminoacyl-tRNA to ribosome.
EF-G is required for translocation
Termination occurs at any one of the three special codons, UAA, UAG, and UGA.
Gene expression may be modulated at the level of translation
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Translation part 2
1. PROTEIN TRANSLATION
From Gene To Protein
ELONGATION AND TERMINATION
HUDA ABBASI
PhD SCHOLAR
NATIONAL UNIVERSITY OF SCIENCES AND TECHNOLOGY
ISLAMABAD, PAKISTAN
2. Three Stages of Translation
1.
INITIATION
Small subunit on
mRNA binding site
is joined by large
subunit
Aminoacyl tRNA
binds
2.
ELONGATION
Ribosome moves
along mRNA
extending protein
by shifting from
peptidyl tRNA to
aminoacyl tRNA
3.
TERMINATION
Polypeptide chain is
released from tRNA
and Ribosome
dissociates from
mRNA
3. 2. ELONGATION
• Elongation includes all the reactions from formation of the
first peptide bond to addition of the last amino acid.
• During elongation, the mRNA moves through the ribosome
and is translated in nucleotide triplets
• Amino acids are added to the chain one at a time.
• Elongation proceeds until a stop codon is reached
• This is the most rapid step of translation process.
4. a. Addition of second tRNA is mediated by
Elongation factor
• Any aminoacyl-tRNA except the initiator can
enter the A site.
• The entry is mediated by an elongation factor
EF-Tu.
• EF-Tu is a highly conserved protein throughout
bacteria and mitochondria and is homologous
to its eukaryotic counterpart.
5. b. EF-Tu is rapidly associated and
disassociated from ribosome
• EF-Tu is associated with the ribosome only
during the process of aminoacyl-tRNA entry.
• Once the aminoacyl-tRNA is in place, EF-Tu
leaves the ribosome to work again with
another aminoacyl-tRNA.
• It displays the cyclic association with, and
dissociation from, the ribosome that is the
hallmark of the accessory factors.
6. • Energy for EF-Tu association and disassociation comes from GTP.
• EF-tu is a GTP binding protein.
• EF-Tu bound to GTP is active
• EF-Tu bound to GDP is inactive
Ts is required to
mediate the
replacement of GDP
by GTP.
(Aminoacyl-tRNA-EF-Tu-GTP)
7. Aminoacyl-tRNA is loaded into the A site
in two stages
• First, the anticodon end binds to the A site of
the 30S subunit.
• tRNA-EF-Tu-GTP [loading complex]
• Second, codon-anticodon recognition triggers
a conformational change in the ribosome.
• This stabilizes tRNA binding and causes EF-Tu
to hydrolyze its GTP.
• EF-Tu-GDP is released, leaving the tRNA on
ribosome.
8. c. First peptide bond is catalyzed by
peptidyl transferase
• Peptide bond formation takes
place by a reaction between the
polypeptide of peptidyl-tRNA in
the P site and the amino acid of
aminoacyl-tRNA in the A site.
• 50S subunit of ribosome has
catalytic property.
10. d. Translocation makes space for third
tRNA
• It is an intrinsic property
of ribosome.
• Translocation expels the
tRNA from the P site to E
site allowing the new
peptidyl-tRNA to enter.
• This makes the A site
ready for entry of the
aminoacyl-tRNA
corresponding to the next
codon.
13. 3. TERMINATION
• Termination encompasses the steps that are
needed to release the completed polypeptide
chain.
• It also includes the dissociation of ribosome the
mRNA.
• At termination the polypeptide and mRNA are
released, the ribosomal subunits dissociate and
are recylced.
What happens to the mRNA? Is it
reused?
14. a. Three codons terminate translation
• 3 out of 64 codons specify termination of
translation
• Stop codons
– UAG (Amber)
– UGA (Opal)
– UAA (Ochre)
15. • Two stages are involved in ending translation.
1. The termination reaction involves release of
the protein chain from the last tRNA.
2. The post-termination reaction involves
release of the tRNA and mRNA and
dissociation of the ribosome into its subunits.
16. b. Stop codons are recognized by release
factors
• Class I and class II release factors
• Termination codons are recognized by class I release factors
(RF 1 & 2).
– RF1 recognizes UAA and UAG
– RF2 recognizes UAA and UGA.
• The factors act at the ribosomal A site and require poly
peptidyl-tRNA in the P site.
• Class II release factor (RF3) releases class I RFs from
ribosome.
17. • binding of release factor
to a stop codon
terminates translation
• the completed
polypeptide is released
• the ribosome dissociates
into its two separate
subunits
19. At stop codon no tRNA can fill the empty A site
of the elongation complex.
Instead the A site is filled by a release factor, and
the amino acid is cleaved from the tRNA that
occupies the P site of the ribosome.
20. c. Termination is chemically similar to
elongation
Release factor at A
site transfers a
hydroxyl group to
P site
Aminoacyl-tRNA at
A site attacks C-O
bond at P site
Peptide chain is
released
Peptide chain is
attached to tRNA
on A site
21. Conclusion of termination
• After release factor (RF),
ribosome recycling factor
(RRF) binds at A site.
• RRFs mediate dissociation
of ribosomal subunits.
22. Accuracy of translation
• One error for every 104 to 105 amino acids
incorporated.
The ribosome can make two types of errors in
translation.
• It may cause a frame-shift by skipping a base
when it reads the mRNA
OR
• by reading a base twice-once as the last base of
one codon and then again as the first base of
the next codon, or twice within the same
codon.
23. An aminoacyl-tRNA synthetase can make two
types of errors:
• It can place the wrong amino acid on its tRNA
Or
• It can charge its amino acid with the wrong
tRNA
25. Concept 1: How does a ribosome recognize only the tRNA
corresponding to the codon in the A site?
A. Any substrate initially can contact the active center by a random-hit
process, but then the wrong substrates are rejected and only the
appropriate one is accepted.
Concept 2: How do tRNAs recognize the corresponding amino
acids?
A. The structures of each amino acid are different, and those differences
generate one half of the specificity of the synthetase. In addition, the
structure of each tRNA is also different, which provides the other half of
the specificity.
Common concepts of translation
27. REGULATION OF TRANSLATION
Example: LAC Operon repressor protein
- Repressor protein is continuously transcribed but only 10
tetramers are present in a cell at any given time
28. REGULATION OF TRANSLATION
A regulator protein may block translation by binding to a site on mRNA
that overlaps the ribosome binding site at the initiation codon.
29. Natural translation inhibitors
• Kirromycin inhibits the function of EF-Tu.
The EF-Tu-GDP complex cannot be released from the ribosome .
As a result, the ribosome becomes "stalled" on mRNA, bringing
translation to a halt.
• Puromycin induces premature termination
Puromycin resembles an amino acid attached to the tRNA. It
adds to the growing polypeptide inhibiting the addition of
further amino acids. This premature termination of translation is
responsible for the lethal action of the antibiotic.
30. Commercial translation inhibitors
• Chloramphenicol
• Erythromycin
• Clindamycin
Etc…
How do antibiotics target only
bacterial protein synthesis and
not ours?
31. Summary
Elongation factors are required to add amino
acids to polypeptide chain.
EF-Tu binds aminoacyl-tRNA to ribosome.
EF-G is required for translocation
Termination occurs at any one of the three
special codons, UAA, UAG, and UGA.
Gene expression may be modulated at the
level of translation