Eukaryotic Translation & its Regulation

1. WELCOME

3. What are Eukaryotes ?
 Eukaryotes are
organisms with a
complex cell or cells,
in which the genetic
material is organised
into a membrane
bound nucleus or
nuclei and it also
contains cell
orgenelles.

4. What are Translation ?
• Translation is basically a synonym process of
protein synthesis.
• Translation is the process by which protein is
synthesized from the information contained in
a molecule of messenger RNA (mRNA).
• It can defined as “the process by which the
sequence of nucleotides in a messenger RNA
molecule directs the incorporation of amino
acid into protein”.

5. Translation : An Overview
• Ribosomes translate the genetic message of
mRNA into proteins.
• The mRNA is translated from 5` 3`.
• Amino acids bound to tRNA are inserted in a
proper sequence due to :
Specific binding of each amino acid to its
tRNA.
Specific base-pairing between the mRNA
codon and tRNA anticodon.

6. Components of Translation
 mRNA :
- Made in the nucleus, transported to cytoplasm.
 tRNA :
- Adaptor molecule that mediate the transfer of information form
nucleic acid to protein.
 Ribosomes :
- Manufacturing units of a cell.
 Enzymes :
- Required for the attachment of amino acids to the correct tRNA
molecule, and for peptide bond formation between amino acids.
 Proteins :
- Soluble factors necessary for proper initiation, elongation and
termination.

7. Enzymes
• Aminoacyle-tRNA Synthetases catalyze the
attachment of tRNA molecule to its respective
amino acid.
- At least one for each tRNA.
- Attachment of amino acid activates/changes the
tRNA molecule.
• Peptidyl Transferase
- Forms the peptide bond between the amino
acids.

8. TRANSLATION MACHINERY
• The machinery required for translating the language of messenger RNAs
into the language of proteins is composed of four primary components.
• mRNA : Messenger RNA (mRNA) provides an intermediate that carries
the copy of a DNA sequence that represents protein.
• tRNA : tRNA acts as an adaptor between the codons and the amino acids
they specify.
• Enzymes : Required for the attachments of amino acids to the correct
tRNA molecule.
a. Aminoacyle-tRNA Synthetases.
- Attachment of amino acid charges the tRNA molecule.
b. Peptidyl Transferase.
- Forms the peptide bond between the amino acids.
• Ribosome : It is the macromolecular complex that directs the synthesis of
proteins.

9. TRANSLATION PROCESS
• In a prokaryotic cell, transcription and translation
are coupled; that is, translation begins while the
mRNA is still being synthesized. In a eukaryotic
cell, transcription occures in the nucleus, and
translation occurs in the cytoplasm.
• Translation involves three major steps :
1. INITIATION
2. ELONGATION
3. TERMINATION

10. 1. INITIATION
• The initiation of translation in eukaryotes is
complex, involving at least 10 eukaryotic
initiation factors (eIFs) & divided into 4 steps :
a. Ribosomal dissociation.
b. Formation of 43S preinitition complex.
c. Formation of 48S initiation complex.
d. Formation of 80S initiation complex.

11. a. Ribosomal dissociation
• The 80S ribosome
dissociates to form 40S &
60S subunits.
• Two initiating factors
namely eIF-3 & eIF-1A
bind to the newly formed
40S subunit & thereby
block its reassociation
with 60S subunit.

12. b. Formation of 43S preinitition
complex.
• A ternery complex
containing met-tRNA’
& eIF-2 bound to GTP
attaches to 40S
ribosomal subunit to
form 43S preinitiation
complex.
• The presence of eIF-3
& eIF-1A stabilizes
this complex.

13. c. Formation of 48S initiation
complex.
• The binding of mRNA to
43S preinitiation complex
results in the formation of
48S initiation complex
through the intermediate
43S initiation complex.
• eIF-4F complex is formed
by the association of eIF-
4G, eIF-4A with eIF-4E.
• The eIF-4F (referred to as
cap binding protein) binds
to the cap of mRNA.

14. • Then eIF-4A & eIF-4B bind to mRNA & reduce
its complex stucture.
• This mRNA is then transferred to 43S complex.
• For the appropriate association of 43S
preinitiation complex with mRNA, energy has to
be supplied by ATP.
• The ribosomal initiation complex scans the
mRNA for the identification of appropriate
initiation codon.
• 5’-AUG is the initiation codon.

15. d. Formation of 80S initiation
complex.
• 48S initiation complex binds to
60S ribosomal subunit to form
80S initiation complex.
• The binding involves the
hydrolysis of GTP (bound to
eIF-2).
• This step is facillatated by the
involvement of eIF-5.
• As the 80S complex is formed,
the initiation factors bound to
48S initiation complex are
released & recycled.

16. 2. ELONGATION
• Ribosomes elongate the polypeptide chain by a
sequential addition of amino acids.
• The amino acids sequence is determined by the
order of the codons in the specific mRNA.
• Elongation, a cyclic process involving certain
elongation factors (Efs).
• Elongation may be divided into three steps.
a. Binding of Aminoacyl t-RNA to A-site.
b. Peptide bond formation.
c. Translocation.

17. a. Binding of Aminoacyl t-RNA to A-
site
• The 80S initiation complex contains met tRNA in
the P-site & A-site is free.
• Another Aminoacyl-tRNA is placed in the A-site.
• This requires proper codon recognization on the
mRNA & involvement of elongation factor
1a(EF-1a) & supply of energy by GTP.
• The aminoacyl t-RNA is placed in the A-site,EF-
1a & GDP are recycled to bring another
Aminoacyl-tRNA.

18. b. Peptide bond formation
• The enzyme Peptide transferase catalyzes the
formation of peptide bond.
• The activity of this enzyme lies on 28S RNA of
60S ribosomal subunit.
• It is therefore the rRNA (and not protein) referred
to as ribozyme that catalyzes peptide bond
formation.
• Net result of peptide bond formation is the
attachment of the growing peptide chain to the
tRNA in the A-site.

19. c. Translocation
• The ribosome moves to the next codon of the mRNA.
• This process called translocation, involves the
movement of growing peptide chain from A-site to P-
site.
• Translocation requires EF-2 & GTP.
• GTP gets hydrolyzed and supplies energy to move
mRNA.
• EF-2 & GTP complex recycles for translocation.
• About six amino acids per second are incorporated
during the course of elongation of translation in
eukaryotes.

20. Step 1 : Decoding
Elongation Cycle
Step 2 : Peptide bond
formation
Step 3 :
Translocation

21. 3. TERMINATION
• One of the stop or termination signals(UAA, UAG and UGA)
terminates the growing polypeptide.
• When the ribosome encounters a stop codon,
- there is no tRNA available to bind to the A site of the ribosome,
- instead a release factor binds to it.
• In eukaryotes, a single release factor-eukaryotic release factor 1
(eRF-1)-recognizes all three stop codons, and eRF-3 stimulates the
termination events.
• Once the release factor binds, the ribosomes unit falls apart,
- releasing the large and small subunits,
- the tRNA carrying the polypeptide is also released, freeing up the
polypeptide product.
• Ribosome recycling occures in eukaryotes.

22. Regulation by RNA binding protein
• Inhibitory effect on translation except PABP.
• For translation these protein must be degraded.

23. Regulation by specific 5’ UTR
protein interaction :
• Rare but just one example : ferritin mRNA (iron).
• For strong inhibition of translation requires the
protein RNA interaction at cap proximal location,
prevent loading of 43S complex onto mRNA.
• Inhibition is much weaker if protein binding RNA
motif moved to more cap distal position.
• In this 43S complex loaded over the mRNA, it’s
subsequent scanning will displace the bound
protein.

24. Stimulation by 3’ poly tail :
• PABP have stimulatory effect.
• PABPs second RRM domain interact with
eIF4G, result in the circulation of mRNA in
the closed loop configuration.
• By the help of PABP eIF4F remain anchor to
mRNA at 3’ poly A tail, this anchoring will not
be occure in the absense of PABP or poly A
tail.

25. Regulation by specific 3’ UTR
protein interaction :
• Control by 3’UTR is entirely dependent on
changes in poly A tail length, because the
regulatory mRNA are repressed when they
have a short tail or activated when they have a
long length.
• In some cases translation can be activated with
out the lengthening of short poly A tail, in
some cases protein may changes the
polyadenylation status.

26. Translation regulation by miRNAs :
• Sequence specific.
• Repress translation at 3’UTR
• Can act in conjunction with RNA binding protein
• Almost 21 nucleotide
• Degree of repression increases with the increasing
number of miRNA
• Repression efficiency might also be influenced by the
distance and sequence between miRNA target sites and
also their position in the 3’UTR
• In some cases miRNA act as a adaptor for sequence
specific RNA binding protein.

27. • The mechanism of repression have 2 main
component
- Normal mRNA translation
- Deadenylation dependent pathway
• Repressed mRNA displace from large
polysome to small polysome or sub polysomal
partical, this indicate the inhibition of
initiation.

28. Phosphorylation of eukaryotic translation
initiation factor eIF4E contributes to its
transformation and mRNA transport activities
• The eukaryotic translation initiation factor eIF4E
is dysregulated in a wide variety of human
cancers.
• In the cytoplasm, eIF4E acts in the rate-limiting
step of translation initiation whereas in the
nucleus, eIF4E forms under nuclear bodies.
• Thus, phosphorylation of nuclear eIF4E seems to
be an important step in control of the mRNA
transport and thus the transforming properties of
eIF4E.
Topisirovic et al. (2004)

29. Controlling gene expression through RNA regulations :
The role of the eukaryotic translation initiation factors
eIF4E
• The eukaryotic translation initiation factor eIF4E is a potent
oncogene.
• In fact its overexpression in human cancer often correlates with poor
progenosis.
• Additionally, eIF4E may play a role in mRNA sequestration and
stability in cytoplasmic processing bodies.
• Hence, eIF4E functions as a central node of an RNA regulation,
which plays an essential role in normal differentiation and
development and is frequently dysregulated in cancer.
• Here, the physiological implications of these observations are
described and the clinical implications of directly targeting eIF4E,
and related regulon are discussed.
Culikovic et al. (2007)

30. Source:
 Genome 4 by T. A. Brown
 Topisirovic I. (2004). Phosphorylation of eukaryotic translation
initiation factor eIF4E contributes to its transformation and
mRNA transport activities.64: 8639-8642.
 Culjkovic B. (2007). Controlling gene expression through RNA
regulations : The role of the eukaryotic translation initiation
factors eIF4E. 6:65-69.