Transcript Document

Regulation of Protein Translation
By Emmanuel Landau
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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.
There are two overall mechanisms of
translation control.
1. Regulation by modifying proteins
2. Regulation using micro RNAs
(miRNA’s)
Mechanism of Translation
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
RNA’s and 78 different proteins.
It is organized in two subunits:
A 40S and a 60S subunit.
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.
Sequence of events
leading to
translation initiation
Sonenberg et al., eds., Translational Control of
Gene Expression (2000), p. 46.
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 eIF4’s (G,A,B,E) are collectively
known as eIF4F.
The Binding of eIF4F to the 5’ Cap of mRNA
Modified from Sonenberg et al., eds. Translational Control of Gene Expression (2000), p. 46.
Regulation of Step 1
1. Phosphorylation of the eIF4E
binding proteins, the 4E-BPs.
Why does this help?
Because the 4E-BPs inhibit
eIF4E’s function, and their
phosphorylation liberates
eIF4E from inhibition.
Phosphorylation of 4EBP Allows eIF4E
Association with eIF4G
Sonenberg et al. eds Translational Control of Gene Expression (2000) p. 247
Phosphorylation of 4EBP by mTOR
and Upstream Pathway
Sonenberg et al. eds Translational Control of Gene Expression (2000) p. 252
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.
Polyadenylation and Circularization of mRNA
Through Binding of PABP to eIF4G
Lodish et al. Molecular Cell Biology Fig. 4-42
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
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 capbinding complex.
Polyadenylation Leads to the PABP-mediated
Displacement of Maskin from eIF4E
Modified from Groisman et al. Cell 109: 473 (2002)
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.
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
Initiation Step 2: Assembly of the
Preinitiation Complex
1. Initiation factors 1, 1A, and 3
bind to the 40S ribosomal subunit.
2. 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.
3. mRNA-eIF4F now binds to 40S
via eIF3.
Assembly of the Preinitiation Complex
Modified from Sonenberg et al., eds. Translational Control of Gene Expression (2000), p. 46.
Initiation Step 3: mRNA Scanning,
AUG Recognition and Ribosome
Completion (40S+60S=80S)
1. The preinitiation complex travels
downstream along the 5’UTR of the
mRNA until it arrives at the start
codon (AUG) and recognizes it
through interaction with the eIF2GTP-tRNA complex.
2. GTP is hydrolyzed by eIF5, the
preinitiation complex unravels, the 60S
subunit binds to the 40S subunit and
translation begins.
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.
The Elongation Cycle (1)
1. Translation starts with the AUG start
codon positioned at the A site of the
ribosome.
2. As the ribosome continues to scan the
mRNA, the AUG-tRNA-Met complex
is translocated to the P site of the
ribosome.
3. A new AA-tRNA arrives at the vacated
A site, courtesy of elongation factor 1A
(eEF1A).
4. eEF1A requires GTP for activation. Its
GEF is eEF1B.
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 9:160 (2003).
Elongation Cycle (2a)
1. 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.
2. 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.
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. 29:25 (2004)
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.
Binding of eEF2 • GTP to Ribosome Catalyzes tRNA
Translocation from Small Subunit Binding Sites
EF-G is Bacterial Homologue of eEF2
Joseph, RNA 9:160 (2003).
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).
GPCRs
Protein Kinase A
NMDA-R
TRK-B
MAP Kinase
PKC
mTOR - p70-S6K
Translation Pathway
4E-BP
Rapamycin
Proteins Involved
in Translation
5’ TOP
mRNAs
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.
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 100:311 (2000).
Conclusions
Translation is regulated at all 3 phases: initiation, elongation,
and termination.
Initiation 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.