Title: Regulation of Protein Translation
1Regulation of Protein Translation
- By Emmanuel Landau
- For other ScienceMag teaching resources see
- http//stke.sciencemag.org/resources/education/arc
hive.dtl
2Why Regulate Translation?
- Translation
- Produces proteins rapidly Produces
proteins locally - Produces single proteins
- or classes of proteins
- BUT
- It is very costly in energy.
3There are two overall mechanisms of translation
control.
- Regulation by modifying proteins
- Regulation using micro RNAs (miRNAs)
4Mechanism of Translation
5Translation 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.
6Phases 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.
7Sequence of events leading to translation
initiation
Sonenberg et al., eds., Translational Control
of Gene Expression (2000), p. 46.
8Initiation 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.
9The Binding of eIF4F to the 5 Cap of mRNA
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
10Regulation 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.
11Phosphorylation of 4EBP Allows eIF4E Association
with eIF4G
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 247
12Phosphorylation of 4EBP by mTOR and Upstream
Pathway
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 252
13Regulation 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.
14Polyadenylation and Circularization of mRNA
Through Binding of PABP to eIF4G
Lodish et al. Molecular Cell Biology Fig. 4-42
15Circular 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
16In 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.
17Polyadenylation Leads to the PABP-mediated
Displacement of Maskin from eIF4E
Modified from Groisman et al. Cell 109 473 (2002)
18Regulation 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.
19MAPK-Dependent Phosphorylation of eIF4E Is
Mediated by the eIF4G Associated Kinase Mnk
Sonenberg et al. eds Translational Control of
Gene Expression (2000) p. 270
20Initiation 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.
21Assembly of the Preinitiation Complex
Modified from Sonenberg et al., eds.
Translational Control of Gene Expression (2000),
p. 46.
22Initiation 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.
23Disassembly 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.
24The 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.
25Elongation 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).
26Elongation 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.
27Elongation 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)
28The 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.
29Binding 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).
31mTOR - p70-S6K Translation Pathway
4E-BP
Rapamycin
5 TOP mRNAs
32Termination 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.
33Mimicry 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).
34Conclusions
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.