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Regulation of Protein Translation

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Title: Regulation of Protein Translation


1
Regulation of Protein Translation
  • By Emmanuel Landau
  • For other ScienceMag teaching resources see
  • http//stke.sciencemag.org/resources/education/arc
    hive.dtl

2
Why Regulate Translation?
  • Translation
  • Produces proteins rapidly Produces
    proteins locally
  • Produces single proteins
  • or classes of proteins
  • BUT
  • It is very costly in energy.

3
There are two overall mechanisms of translation
control.
  • Regulation by modifying proteins
  • Regulation using micro RNAs (miRNAs)

4
Mechanism of Translation
5
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.

6
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.

7
Sequence of events leading to translation
initiation
Sonenberg et al., eds., Translational Control
of Gene Expression (2000), p. 46.
8
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.

9
The Binding of eIF4F to the 5 Cap of mRNA
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
10
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.

11
Phosphorylation of 4EBP Allows eIF4E Association
with eIF4G
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 247
12
Phosphorylation of 4EBP by mTOR and Upstream
Pathway
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 252
13
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.

14
Polyadenylation and Circularization of mRNA
Through Binding of PABP to eIF4G
Lodish et al. Molecular Cell Biology Fig. 4-42
15
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
16
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.
17
Polyadenylation Leads to the PABP-mediated
Displacement of Maskin from eIF4E
Modified from Groisman et al. Cell 109 473 (2002)
18
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.

19
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
20
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.

21
Assembly of the Preinitiation Complex
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
22
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.

23
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.
24
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.

25
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).
26
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.

27
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)
28
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.

29
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).
30
  • 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).

31
mTOR - p70-S6K Translation Pathway
4E-BP
Rapamycin
5 TOP mRNAs
32
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.

33
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).
34
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.
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