Regulation of Protein Translation

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Regulation of Protein Translation

• By Emmanuel Landau
• For other ScienceMag teaching resources see
• http//stke.sciencemag.org/resources/education/arc
  hive.dtl

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Why Regulate Translation?

• Translation
• Produces proteins rapidly Produces
  proteins locally
• Produces single proteins
• or classes of proteins
• BUT
• It is very costly in energy.

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There are two overall mechanisms of translation
control.

• Regulation by modifying proteins
• Regulation using micro RNAs (miRNAs)

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Mechanism of Translation
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Translation generates proteins according to the
instructions read from messenger RNA (mRNA).

• mRNA is translated by moving through a groove in
  the ribosome. The ribosome is a multicomponent
  entity, composed of ribosomal RNAs and 78
  different proteins.
• It is organized in two subunits
• A 40S and a 60S subunit.

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Phases of Translation

• Translation is divided into two stages
  initiation and elongation.
• Initiation brings the mRNA to the ribosome and
  uses a large number of initiation factors to
  assemble the ribosome and begin translation.
• Elongation then continues to assemble amino acids
  to form the protein.

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Sequence of events leading to translation
initiation
Sonenberg et al., eds., Translational Control
of Gene Expression (2000), p. 46.
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Initiation Step 1. Capture of mRNA

• The 5 cap (m7GpppX) of the mRNA is captured by
  binding to a complex of eukaryotic Initiation
  Factors of the eIF4 family.
• These are eIF4G, a scaffold protein eIF4E, which
  is bound to eIF4G and holds the mRNA and eIF4A
  and eIF4B, which unravel kinks in the mRNA.
• The four eIF4s (G,A,B,E) are collectively known
  as eIF4F.

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The Binding of eIF4F to the 5 Cap of mRNA
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
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Regulation of Step 1

• 1. Phosphorylation of the eIF4E
  binding proteins, the 4E-BPs.
• Why does this help?
• Because the 4E-BPs inhibit eIF4Es
  function, and their phosphorylation liberates
  eIF4E from inhibition.

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Phosphorylation of 4EBP Allows eIF4E Association
with eIF4G
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 247
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Phosphorylation of 4EBP by mTOR and Upstream
Pathway
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 252
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Regulation of Step 1

• 1. Phosphorylation of the eIF4E
  binding proteins, the 4E-BPs.
• 2. Binding of polyadenylate binding protein
  (PABP) to eIF4G.
• Why?
• Because this circularizes the polysome, and
  allows ribosomal subunits to start new ribosomes.

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Polyadenylation and Circularization of mRNA
Through Binding of PABP to eIF4G
Lodish et al. Molecular Cell Biology Fig. 4-42
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Circular mRNA Visualized by Atomic Force
Microscopy
eIF4E, eIF4G, and PABP are Present in the
Light-Colored Regions Attached to each RNA
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 454
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In case of mRNAs with a CPE sequence in the 3
end, the poly(A) tail also serves to disrupt the
binding of maskin, a CPEB-binding protein to
eIF4E. This makes eIF4E available to start
building the cap- binding complex.
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Polyadenylation Leads to the PABP-mediated
Displacement of Maskin from eIF4E
Modified from Groisman et al. Cell 109 473 (2002)
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Regulation of Step 1

• 1. Phosphorylation of the eIF4E
  binding proteins, the 4E-BPs.
• 2. Binding of polyadenylate binding protein
  (PABP) to eIF4G.
• 3. Phosphorylation of eIF4E allows it to detach
  from the cap and recycle.

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MAPK-Dependent Phosphorylation of eIF4E Is
Mediated by the eIF4G Associated Kinase Mnk
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 270
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Initiation Step 2 Assembly of the Preinitiation
Complex

• Initiation factors 1, 1A, and 3 bind to the 40S
  ribosomal subunit.
• eIF2, activated by GTP and carrying Met-tRNA,
  joins the 40S complex. GDP-GTP exchange on eIF2
  is enhanced by eIF2B, which acts as a GEF. eIF2
  is inhibited by direct phosphorylation.
• mRNA-eIF4F now binds to 40S via eIF3.

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Assembly of the Preinitiation Complex
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
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Initiation Step 3 mRNA Scanning, AUG Recognition
and Ribosome Completion (40S60S80S)

• The preinitiation complex travels downstream
  along the 5UTR of the mRNA until it arrives at
  the start codon (AUG) and recognizes it through
  interaction with the eIF2-GTP-tRNA complex.
• GTP is hydrolyzed by eIF5, the preinitiation
  complex unravels, the 60S subunit binds to the
  40S subunit and translation begins.

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Disassembly of the Preinitiation Complex Upon
Recognition of the Start Codon (AUG)
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
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The Elongation Cycle (1)

• Translation starts with the AUG start codon
  positioned at the A site of the ribosome.
• As the ribosome continues to scan the mRNA, the
  AUG-tRNA-Met complex is translocated to the P
  site of the ribosome.
• A new AA-tRNA arrives at the vacated A site,
  courtesy of elongation factor 1A (eEF1A).
• eEF1A requires GTP for activation. Its GEF is
  eEF1B.

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Elongation Sequence of tRNA Displacements (A P
E Sites) And Crystal Structures of the Ribosome
Subunits Bacterial 30S and 50S Subunits of the
Bacterial Ribosome are Shown, Complexed with EF-G
(homologous to Eukaryotic 40S, 60S, and eEF2)
Joseph, RNA 9160 (2003).
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Elongation Cycle (2a)

• The AUG-tRNA-met is now at the P site. The next
  available codon of the mRNA is now at the A site
  and the cognate aa-tRNA-eEF1A-GTP binds to it.
• The ribosome catalyzes a peptide bond between
  the methionine at the P site and the new amino
  acid. However, its tRNA is still at the A site.

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Elongation Successive AAs are Brought to the
Vacant A-Site by Cognate tRNA Bound to eEF1A
GTP (E-Site not Shown Nascent Protein at P-Site
tRNA)
Abbott and Proud, Trends Biochem.Sci. 2925 (2004)
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The Elongation Cycle (2b)

• 3. Elongation factor eEF2-GTP enters the
  ribosome, pushing the new tRNA into the P site,
  and the deacylated first tRNA into the exit (E)
  site. In the next cycle this tRNA will be
  ejected from the ribosome.
• 4. During this process GTP is hydrolyzed, and
  eEF2-GDP leaves the ribosome.
• 5. The cycle is repeated many times.

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Binding of eEF2 GTP to Ribosome Catalyzes tRNA
Translocation from Small Subunit Binding
Sites EF-G is Bacterial Homologue of eEF2
Joseph, RNA 9160 (2003).
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• Translation can also be regulated by controlling
  the synthesis of translation factors.
• This process is mediated by the Ser/Thr kinase
  mTOR (mammalian target of rapamycin).

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mTOR - p70-S6K Translation Pathway
4E-BP
Rapamycin
5 TOP mRNAs
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Termination of Translation

• Releasing Factors (eRFs) are involved in
  termination.
• eRF1 structurally mimics tRNA that is bound to
  eEF1a GTP. eRF1 fits into the ribosomal
  A-site, where it recognizes the stop codon. It
  then releases the completed polypeptide by
  catalyzing a nucleophilic attack on the ester
  bond between the peptide and the P-site tRNA.
• The catalytic activity of eRF1 is stimulated by
  the GTP-bound form of another relasing factor,
  eRF3.

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Mimicry of tRNA by the Releasing Factor eRF1
Domain 2 on eRF1 Recognizes the Stop Codon Domain
3 Catalyzes the Hydrolysis of the Completed
Peptide from the P-Site tRNA
Song et al., Cell 100311 (2000).
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Conclusions
Translation is regulated at all 3 phases
initiation, elongation, and termination. Initiati
on is the most highly regulated of these phases,
involving a large number of initiation factors
and accessory proteins. In addition to
modification of translation factors either by
phosphorylation or by GDP/GTP exchange, and the
modification of mRNAs at their 3 UTRs, protein
synthesis can be controlled by the mTOR-mediated
synthesis of additional translational machinery.