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The initiation phase of protein synthesis in eukaryotes
The final phase of protein synthesis - Translational Termination
The structure of a human translation release factor (eRF1) and its resemblance to a tRNA molecule
Translational Control
Two Types of Translational Control
1] Global regulation
Two Types of Translational Control
1] Global regulation
2] Message specific regulation
Two Types of Translational Control
• Initiation - Recruitment of the ribosome to the mRNA Recognition of the start codon
• Elongation - Movement of the ribosome along the mRNA Decoding the mRNA into an extending polypeptide chain
• Termination - Recognition of the stop codon Release of the ribosome and protein chain from the mRNA
Review of Eukaryotic mRNA translation
AAAAAAAAAAA
7mGpppN
ORF5’UTR3’UTR
startstop
• Initiation - Recruitment of the ribosome to the mRNA Recognition of the start codon
Review of Eukaryotic mRNA translation
AAAAAAAAAAA
7mGpppN
ORF5’UTR3’UTR
startstop
Initiation Recruitment of ribosome and the initiator methionyl tRNA to the start codon
Initiation Recruitment of ribosome and the initiator methionyl tRNA to the start codon
eIF1AeIF2 eIF2BeIF3eIF4AeIF4BeIF4EeIF4GeIF5eIF5Baminoacylated initiator methionyl tRNA (Met-tRNAi
Met)
40S ribosomal subunit60S ribosomal subunitGTP ATP
Initiation Recruitment of ribosome and the initiator methionyl tRNA to the start codon
Five Steps-
1. 40S ribosomal subunit and tRNAiMet
preparation
2. mRNA selection and preparation
3. 40S/ mRNA binding
4. scanning and AUG recognition
5. 60S ribosomal subunit joining
eIF1AeIF2 eIF2BeIF3eIF4AeIF4BeIF4EeIF4GeIF5eIF5Baminoacylated initiator methionyl tRNA (Met-tRNAi
Met)
40S ribosomal subunit60S ribosomal subunitGTP ATP
60S
40S 40S3
60S
1A3 1A
2GDP
2 GTP
GTP
GDP
Met
2 GTPMet
2B
eIF2•GTP•Met-tRNAiMet is called the
Ternary Complex-TC
3 2 GTPMet
1 1A
1
Step 1: 40S ribosomal subunit and tRNAiMet preparation
Met-tRNAiMet
43S pre-initiation complex
60S
40S 40S3
60S
1A3 1A
2GDP
2 GTP
GTP
GDP
Met
2 GTPMet
2B
eIF2•GTP•Met-tRNAiMet is called the
Ternary Complex-TC
3 2 GTPMet
1 1A
1
Formation of Ternary Complex an important regulatory node
Met-tRNAiMet
43S pre-initiation complex
Step 2: mRNA selection and preparation
11
AUGCap4E 4G
4A4B
• The mRNA is bound by eIF4F complex composed of eIF4E, eIF4G, eIF4A.• eIF4B binds to eIF4A and facilitates the helicase activity of 4A that is required to
unwind secondary structures in the mRNA during scanning
eIF4F
Step 2: mRNA selection and preparation
12
AUGCap4E 4G
4A4B
eIF4FeIF4E binding is an important
regulatory node
3 2 GTPMet
1 1AAUGCap
4E 4G4A4B
43S mRNA interactioneIF3 in the 40S complex and eIF4G in the mRNA complex interact
Step 3: 43S/ mRNA binding and scanning
3 2 GTPMet
1 1A
43S pre-initiation complex
3
AUGCap4E 4G
4A4B
48S pre-initiation complex
AUGCap4E3
2GTPMet
1 1A4G4A4B
Scanning
• The 48S complex scans each codon in a 5’ to 3’ direction looking for an AUG.• The eIF4A helicase activity irons out RNA hairpins allowing the 40S complex to move.• multiple rounds of ATP hydrolysis are required to provide energy for the movement
Step 3: 43S/ mRNA binding and scanning
3 2 GTPMet
1 1ACap
AUG recognition
• eIFs 1, 2 and 5 are required for this step.
AUG
5
5
GTPase step and recycling of eIFs
• eIF5 stimulates the GTPase activity of eIF2 (GTP hydrolysis to GDP) leading to loss of initiation factor binding.
32
Met
1
1ACap AUG
5GDP
Step 4: AUG recognition and GTP hydrolysis
16
Met
Cap AUG
60S
5B
Met
Cap AUG
GTP
GDP
Step 5: 60S Joining
Elongation
GTP
5B GDP
1A
A second G protein eIF5B promotes 60S subunit joining. Hydrolysis of GTP to GDP promotes 5B and eIF1A release. The elongation
phase, where the protein is synthesized can now begin.
1A
Measurement of Translation
Reporter Assays
poly(A)Reporter
luciferaseB-galGFP
Westerns
Extract proteins
Run on SDS-Page
Probe with Antibody
35S incorporation
Pulse-label 35S met or cys
Take time points
Measure incorporation by TCA prec. followed by scintillation
0
1
2
3
4
5
2 4 6 8 100
Time (mins)C
PMin
corp
orat
ed( X
10)
3
Polysome analysis
Sucrose gradient
Yeast Extract
Spin
Collect fractions
28S rRNA
18S rRNA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
rRN
A
Efficiently translatedmRNA
Poorly translatedmRNA
mR
NA
s
mRNA
40S subunit
60S subunit
80S ribosome
polyribosomes
Abs
260n
m
FractionsTop Bottom
Non-translating RNAs
Translation efficiencymRNA
40S
60S
80S
polyribosomes
1] Global regulation
2] Message specific regulation
Two Types of Translational Control
0 min
P
80S
RNP
120 min
GAL-Dhh1pCondition A Condition B
1] Global regulation
0 min
P
80S
RNP
120 min
GAL-Dhh1pCondition A Condition B
1] Global regulation
Usually seen during conditions of stress or stimulation by hormone
Ternary Complex formation
eIF-4E regulation
1] Global regulation
3 2 GTPMet
1 1AAUGCap
4E 4G4A4B
43S mRNA interactioneIF3 in the 40S complex and eIF4G in the mRNA complex interact
Step 3: 43S/ mRNA binding and scanning
3 2 GTPMet
1 1A
43S pre-initiation complex
3
AUGCap4E 4G
4A4B
48S pre-initiation complex
Three Types of eIF4E regulation
1] Transcriptional
2] eIF4E phosphorylation
3]Binding of inhibitory proteins
Transcriptional Regulation of eIF4E
eIF4E is the least abundant initiation factor in cells
Transcription increase several fold in fibroblast upon growth factor treatment
Mechanism poorly understood, but involves c-Myc regulation.
Links to cancer
Phosphorylation of eIF4E
eIF4E phosphorylated at a single site -Ser209
Phosphorylation increases translation rate
Structure of eIF4E with m7GpppG analog
Ser209
m7GpppG
Phosphorylated Ser209 interacts with Lys159 to make a retractable salt bridgeClamping mRNA in the cap-binding slot
Ser209
Lys159
Growth Factor, Hormones, Mitogens, and Cytokines mediate eIF4E phosphorylation
932 GINGRAS, RAUGHT & SONENBERG
TABLE 1 Effect of various stimuli on eIF4E and 4E-BP1 phosphorylation
Stimulus Cell type eIF4E 4E-BP1 References
Adenovirus HeLa, 293 -a +b 227, 228
infection (early)
Angiotensin II Vascular smooth muscle + + 208, 357
Anisomycin Swiss 3T3, 293 + 235
Anti-CD3 Mature CD4+ or CD8+ + 358
thymocytes
Arsenite 293, CHO.K1 + ! 177
Concanavalin A Peripheral blood mono- + 359
nuclear cells
DAMGOc CHO overexpressing + 360
"-receptor
Epidermal growth Mammary + 274
factor epithelial cells
P19 + 361
Swiss 3T3, 3T3-L1, + 362–364
L1, PC12
Gastrin AR4-2J tumor cells + 258
GMCSF + SLF Hematopoietic MO7e + + 365
Heat shock Reuber hepatoma + 231
High glucose Isolated rat pancreatic + 366
islets
Insulin NIH 3T3 + 178, 193, 319
3T3-L1 + + 162, 362, 363
367
CHO + 199, 211
Skeletal muscle + 368, 369
Swiss 3T3; 32D; + 227, 235, 238
293; CHO-IR 370
Gingras, unpublished data). Nevertheless, these data argue that ERKs and p38
act as upstream effectors of eIF4E phosphorylation (Figure 5). It is unclear why
eIF4E phosphorylation is not increased after treatment of cells with sorbitol,
H2O2, or heat shock, which are potent activators of p38 MAP kinases (177).
One hypothesis is that these stresses also decrease phosphorylation of the eIF4E-
binding proteins (see below), thereby reducing the availability of eIF4E for
phosphorylation (177).
(continued)
8505_AR_28 12/2/99 6:44 PM Page 932A
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eIF4 INITIATION FACTORS 933
Insulin-like Rat aortic + 371
growth factor I smooth muscle
Insulin-like Swiss 3T3-L1 + 362, 367
growth factor II
Interleukin 1! CHO.K1 + 177
Interleukin 3 Myeloid progenitor + 238
Lipopolysaccharide B lymphocytes + 212
L-Pyrroline-5- Rabbit reticulocyte + 372
carboxylic acid lysate
Nerve growth factor PC12 + + 194, 364
Platelet-derived NIH 3T3 + 178, 193, 319
growth factor Lung fibroblasts + 373
Swiss 3T3-L1; + 362, 367, 371
aortic SM
PHA + phorbol ester Human T cells + 170
Phorbol ester NIH 3T3, CHO, + 178, 193, 199,
PBL, B cells 211, 212, 319, 359
3T3-L1, + 162, 359, 372
leukaemic T cells,
retic. lysate
Swiss 3T3 + 235
Serum NIH 3T3 + + 178, 193, 256, 319
Swiss 3T3 + 374
CHO + 211
3T3-L1 + 362, 367
Tumor necrosis U937, HeLa, + 177, 375
factor " ME180, BAEC, FS,
HUVEC
a-, No change in phosphorylationb+, Increase in phosphorylation; blank space indicates not determinedcAbbreviations: DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin; GMCSF, granulocyte-macrophage colony stimulating
factor; SLF, steel factor; PHA, phytohemagglutinin.
TABLE 1 (continued)
Stimulus Cell type eIF4E 4E-BP1 References
An excellent candidate for the eIF4E kinase is the MAP kinase-interacting
protein kinase-1(MNK1; also called MAP kinase signal-integrating kinase).
MNK1 was identified independently by two groups as a substrate for ERK1 and
ERK2 (203, 204) and is activated by both the ERK and the p38 map kinases (203;
the signaling pathways to MNK1 activation are depicted in Figure 5). MNK1
phosphorylates eIF4E in vitro on Ser209 (203, 204). Recent experiments strongly
8505_AR_28 12/2/99 6:44 PM Page 933
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Earlier reports suggested that the protein kinase C (PKC) is the physiological
kinase responsible for eIF4E phosphorylation (reviewed in 206), and indeed
PKC (a mixture of !, ", and # isoforms) phosphorylates eIF4E on Ser209 in
vitro (186). Consistent with this hypothesis, insulin-induced phosphorylation of
eIF4E in 3T3-L1 adipocytes (and angiotensin-II–induced phosphorylation in
smooth-muscle cells) is significantly reduced by long-term exposure to phorbol
esters, which downregulate most PKC isoforms (162, 207, 208). Moreover, coin-
jection of PKC and eIF4E potentiates the mitogenic activity of eIF4E in quies-
cent 3T3 cells (209). In other instances, however, no correlation between the
activation of PKC and eIF4E phosphorylation has been observed. For example,
after desensitization of PC12 cells with phorbol esters, nerve growth factor-
induced phosphorylation of eIF4E is unaffected (194). However, chronic treat-
ment with phorbol ester does not affect all PKC isoforms: atypical PKCs, such
as PKC$, are insensitive (210). In CHO cells stimulated with serum or insulin,
eIF4E phosphorylation was not prevented by pretreatment with the PKC inhibitor
eIF4 INITIATION FACTORS 935
Figure 5 Signaling pathways to eIF4E phosphorylation. The intracellular signaling
pathways leading to eIF4E phosphorylation are diagrammed. Also indicated are the
inhibitors (in italics) used to delineate the pathways. Both MNK1 and eIF4E interact
with eIF4G, bringing the two proteins in close proximity, resulting in more efficient
eIF4E phosphorylation.
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Signal Transduction Pathways Mediate eIF4E Phosphorylation
MNK1= MAP kinase-interacting protein kinase
eIF4E interacting proteins
eIF4E binding proteins exist in higher eukaryotes (4E-BP)
Three found in mammals (4E-BP1, 4E-BP2, and 4E-BP3)
Small (10-12 kDa), heat-stable, all contain eIF4E binding site
Binding of 4E-BP does not alter eIF-4E affinity for the cap
B
H A-plY2n) -*
i ;jS T ,43iP
i
J1-F F
- ± + t + -4 - SIT-41;-B']4 - .4fi)
- - + t - + H1A\-P2)
+ + + ± - - -+Il--4 _
- --
1 2 3 45<6 7
Fig. 3. 4E-BP1 and p220 compete for binding to eIF-4E. (A) m7GDP-coupled agarose resin was incubated with buffer A (lanes 1 and 2) or 0.5 jg
recombinant murine eIF-4E in buffer A. The resin was washed in buffer A (3X ml) and then incubated with either buffer A or 3 jig GST-4E-BPl
for 60 min at 4° C. The resin was washed in buffer A (3x 1 ml) and incubated further with buffer A or 50 j1 (-5x 105 cells) of an HA-p220-
expressing SJ9 cell lysate for 60 min at 4° C. The resin was rinsed and bound proteins were eluted in SDS-sample buffer. Proteins were resolved on
SDS-polyacrylamide gels and immunoblotting was performed as described in Materials and methods. (B) As in (A), but m7GDP-bound eIF-4E was
pre-incubated with 100 gl HA-p220-expressing SJ9 cell lysate or 100 ,ul (_I x 106 cells) of uninfected SJ9 cell lysate before further incubation with
1 gg GST-4E-BPI. Incubations periods were as in (A). Minus signs indicate incubation with buffer A.
BPI. Taken together, these findings demonstrate that thebinding of p220 and 4E-BPI to eIF-4E is mutuallyexclusive.
Because the interaction of p220 and 4E-BP1 with eIF-4E was measured on a cap column, it is possible that theinteraction of eIF-4E with the cap affected the outcome
of the results. To circumvent this problem, we also useda glutathione column to bind GST-4E-BP1. In addition,this experiment was also designed to exclude the possibilitythat 4E-BPI and p220 interact directly. GST-4E-BPIbound to the glutathione-Sepharose column, as determinedby Western blotting using an antibody to 4E-BP1 (Figure4, lane 1). No signal was detected with an anti-HAantibody when a lysate of uninfected SJ9 cells was
incubated with either the resin alone (lane 2) or resin withbound GST-4E-BP1 (lane 3). Similarly, when an HA-p220-expressing SJ9 cell lysate was incubated with eitherthe resin alone (lane 4) or resin containing GST-4E-BPI (lane 5), no HA-p220 was retained. This resultdemonstrates that p220 has no affinity for GST-4E-BPI.As anticipated, eIF-4E did not bind by itself to theglutathione column (lane 6) but interacted with GST-4E-BPI (lane 7). A combination of the HA-p220-containingextract and eIF-4E also failed to bind the resin in theabsence of GST-4E-BP1 (lane 8). Most importantly, whenHA-p220 was pre-incubated with the resin containingeIF-4E already bound to GST-4E-BP1, no HA-p220 was
retained on the resin (lane 9; this experiment was conductedat the same time as that shown in Figure 3 which containsa positive control for HA-p220). Taken together, theseresults and those shown in Figures 2 and 3 demonstratethat eIF-4E exists as a complex with 4E-BPI or p220, butnot with both.
The elF-4F complex precludes the association of4E-BP1Notwithstanding the above results, it is possible that 4E-BP1 associates with eIF-4F in cells. In HeLa cells, eIF-4E exists in two forms: as a slowly sedimenting (<6S)form comprising only the 24 kDa CAP binding protein(CBP), and as part of a more rapidly sedimenting (-8-1OS) complex, eIF-4F (Tahara et al., 1981). In the lightof the results described above, it is predicted that 4E-BPIshould not associate with the eIF-4F complex. To examinethis, HeLa cells were lysed in high salt lysis buffer andsubjected to velocity sedimentation on a 10-40% sucrose
gradient. Catalase (1 IS), run in parallel on a separate
gradient, sedimented at fractions 10 and 11 (Figure 5).eIF-2, which has a sedimentation coefficient of -6S(Konieczny and Safer, 1983), was detected mainly infractions 5 and 6, and serves as another sedimentationmarker in this experiment. The immunoblot analysis ofthe fractions sedimenting slower than 11S revealed thetwo forms of eIF-4E (Tahara et al., 1981): one centeredin fraction 3, and the other at fractions 5-7. The highermolecular weight polypeptides p220 and eIF-4A cosedi-mented with eIF-4E, as expected if they were to beassociated with eIF-4E to form the eIF-4F complex. eIF-4A sediments as a singular protein and as part of the eIF-4F complex (Nielsen and Trachsel, 1988). The trailing ofthis protein into lighter fractions represents the free form.In sharp contrast to the sedimentation of the differentinitiation factors, a Western blot analysis of 4E-BPIrevealed that the protein sedimented at the top of thegradient. No 4E-BP1 cosedimented with eIF-4F in frac-tions 5-7, indicating that 4E-BP1 is precluded from theeIF-4F complex. It is worth noting that some 4E-BPI
5704
A.Haghighat et aL
--I,
Haghighat et al., 1995
4E-BP proteins block eIF4E/eIF4G binding
Note: p220 old nomenclature for eIF4G
938 GINGRAS, RAUGHT & SONENBERG
Figure 6 The cocrystal structure of the 4E-BP1 and eIF4GII peptides bound to
eIF4E. (A) The human 4E-BP1 peptide (orange) and the m7GDP cap analog (green)
bind to opposing regions of eIF4E (blue); (B) The 4E-BP1 peptide binds to the dorsal
convex surface of eIF4E and adopts an extended L-shaped conformation; (C) the
eIF4GII peptide (red) also adopts an extended L-shaped conformation when binding
to the same region of eIF4E; (D) a magnified view of the residues involved in medi-
ating 4E-BP1 (orange) binding to eIF4E (blue); (E) sequence alignment of the eIF4E-
binding sites of several 4E-BPs, eIF4Gs, and yeast Caf20; h = human, d = drosophila.
8505_AR_28 12/3/99 1:26 PM Page 938
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4E-BP proteins block eIF4E/eIF4G binding
eIF4G peptide
4E-BP peptide
Gingras et al., 1999
940 GINGRAS, RAUGHT & SONENBERG
Figure 7 The binding of the 4E-BPs to eIF4E is regulated by phosphorylation. The
4E-BPs and eIF4Gs compete for a common binding site on eIF4E. Various stimuli in-
crease the phosphorylation of the 4E-BPs. Hyperphosphorylated 4E-BPs have a rela-
tively low affinity for eIF4E. Conversely, a decrease in 4E-BP phosphorylation in-
creases the affinity of the 4E-BPs for eIF4E. Free eIF4E interacts with eIF4G to form
the translationally active eIF4F complex. GPCR: G protein coupled receptor.
known presently about its phosphorylation sites or regulation (216). 4E-BP regu-
lation appears to be conserved in Drosophila, where some of the inhibitors reduc-
ing human 4E-BP1 phosphorylation also decrease Drosophila 4E-BP
phosphorylation (M Miron, et al submitted).
Signal Transduction Pathway Mediating 4E-BP1 Phosphorylation
The pathway that mediates 4E-BP1 phosphorylation relays signals from PI3K to
Akt and FKBP12-rapamycin-associated protein/mammalian target of rapamycin
(FRAP/mTOR). The function of these proteins and the delineation of their role in
signaling to 4E-BP1 are briefly described below.
Phosphoinositide 3-Kinase (PI3K) The PI3Ks constitute a large family of lipid
kinases that phosphorylate the hydroxyl group at position 3 of the inositol ring of
phosphoinositides (233). PI3Ks have been implicated in the regulation of a variety
of cellular processes, including cell survival, cell motility, proliferation, and differ-
entiation. PI3Ks have been assigned to several families based on their primary
structure features, mode of regulation, and lipid specificity (reviewed in 233, 234).
In response to extracellular stimuli, the regulatory PI3K subunit is recruited to the
membrane, bringing the catalytic subunit close to its lipid substrates.
Stimulus-induced 4E-BP1 phosphorylation is blocked by low concentra-
tions of the inhibitors wortmannin (100 nM) or LY294002 (5 µM) (235; A-C
8505_AR_28 12/3/99 1:32 PM Page 940
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.4E-BP binding to eIF4E is regulated by phosphorylation
!
!"#$%&'()&*!&
+,-.+,-/01(2!3 34/!'210&*0&
5)678&9,4',&4.&3-'6!3&-)&
!"#$:&; 4)'/!(.!.&(<<4)420&
<-/&2,!&=>&'(+&(*-?2&$@
!"#$%&4.&(1.-&('24A(2!3&*0&
+,-.+,-/01(24-)&-<&42.&
*4)34)B&+/-2!4)&2,/-?B,&
'!11&B/-92,&.4B)(14)B
Activation of eIF4E by two distinct yet re-enforcing mechanisms
Ternary Complex formation
eIF-4E regulation
1] Global regulation
60S
40S 40S3
60S
1A3 1A
2GDP
2 GTP
GTP
GDP
Met
2 GTPMet
2B
eIF2•GTP•Met-tRNAiMet is called the
Ternary Complex-TC
3 2 GTPMet
1 1A
1
Step 1: 40S ribosomal subunit and tRNAiMet preparation
Met-tRNAiMet
43S pre-initiation complex
0 min
P
80S
RNP
120 min
GAL-Dhh1p+AminoAcids
-AminoAcids
Amino acid depletion induces a rapid translational repression events in yeast
Alan Hinnebusch and Colleagues
Despite this, some mRNA are upregulated
ANRV253-MI59-18 ARI 4 August 2005 16:40
Figure 3Model for GCN4 translational control. GCN4 mRNA and reinitiating ribosomes are depicted as inFigure 2. The three subunits of eIF2 and the five subunits of eIF2B are listed in the left panel. Negativeregulatory factors are depicted in red; positive effectors are depicted in green. Following translation ofuORF1, !50% of the 40S ribosomes remain attached to the mRNA and resume scanning. Undernonstarvation conditions, the 40S subunit quickly rebinds the TC and reinitiates at uORF4 because theTC concentration is high. Under amino acid starvation conditions, !50% of the rescanning 40Sribosomes fail to rebind the TC until scanning past uORF4, because the TC concentration is low, andreinitiate at GCN4 instead. The remaining !50% rebind the TC before reaching uORF4, translateuORF4, and dissociate. TC levels are reduced in starved cells owing to phosphorylation of eIF2 byGcn2p, thus converting eIF2 from substrate to inhibitor of the GEF eIF2B and reducing the eIF2-GTPlevel in the cell.
reinitiate at GCN4 in starved cells (Figure 3,right panel). As only a few percent of thereinitiating ribosomes can make it pastuORF4 under repressing conditions, thismodel accounts for the 20- to 50-fold in-ducibility of GCN4 translation.
Genetic evidence indicates that reinitiat-ing ribosomes bypass uORFs 2 to 4 underderepressing conditions because the distance
between uORF1 and uORF4 is not largeenough to insure that they rebind the req-uisite factors required for reinitiation beforereaching uORF4. The key evidence for thisconclusion was that ribosomes were preventedfrom reaching GCN4 by gradually increasingthe separation between uORFs 1 and 4 byinserting spacer sequences. When theuORF1-uORF4 separation is expanded to
414 Hinnebusch
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The Presence of uncharged tRNAs stimulates eIF2α phosphorylation
Non-starvation conditions Starvation conditions
Ternary Complex Levels High Ternary Complex Levels Low
60S
40S 40S3
60S
1A3 1A
2GDP
2 GTP
GTP
GDP
Met
2 GTPMet
2B
eIF2•GTP•Met-tRNAiMet is called the
Ternary Complex-TC
3 2 GTPMet
1 1A
1
Step 1: 40S ribosomal subunit and tRNAiMet preparation
Met-tRNAiMet
43S pre-initiation complex
ANRV253-MI59-18 ARI 4 August 2005 16:40
Gcn2p interacts with the Gcn1p/Gcn20pcomplex in vivo in a manner enhanced byGcn20p and dependent on residues 1–225in the NTD of Gcn2p (62). The Gcn2pNTD is sufficient for binding to a C-terminalsegment of Gcn1p (approximately residues2050–2400), and overexpressing either do-main dissociates the native Gcn1p/Gcn2pcomplex and produces a Gcn! phenotype that
is partly suppressed by overproducing eitherof the full-length proteins (62, 104, 105,157). Mutating Arg-2259 in Gcn1p abolishedcomplex formation with Gcn2p and impairedGcn1p function without affecting ribosomebinding or Gcn20p binding by Gcn1p, thusshowing that the Gcn1p-Gcn2p interactionis crucial. The extreme N-terminal (aminoacids 1–672) and C-terminal (amino acids
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GCN2 kinase is a sensor for uncharged tRNA
0 min
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RNP
120 min
GAL-Dhh1p+AminoAcids
-AminoAcids
Amino acid depletion induces a rapid translational repression events in yeast
Alan Hinnebusch and Colleagues
Despite this, some mRNA are upregulated
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ANRV253-MI59-18 ARI 4 August 2005 16:40
Figure 3Model for GCN4 translational control. GCN4 mRNA and reinitiating ribosomes are depicted as inFigure 2. The three subunits of eIF2 and the five subunits of eIF2B are listed in the left panel. Negativeregulatory factors are depicted in red; positive effectors are depicted in green. Following translation ofuORF1, !50% of the 40S ribosomes remain attached to the mRNA and resume scanning. Undernonstarvation conditions, the 40S subunit quickly rebinds the TC and reinitiates at uORF4 because theTC concentration is high. Under amino acid starvation conditions, !50% of the rescanning 40Sribosomes fail to rebind the TC until scanning past uORF4, because the TC concentration is low, andreinitiate at GCN4 instead. The remaining !50% rebind the TC before reaching uORF4, translateuORF4, and dissociate. TC levels are reduced in starved cells owing to phosphorylation of eIF2 byGcn2p, thus converting eIF2 from substrate to inhibitor of the GEF eIF2B and reducing the eIF2-GTPlevel in the cell.
reinitiate at GCN4 in starved cells (Figure 3,right panel). As only a few percent of thereinitiating ribosomes can make it pastuORF4 under repressing conditions, thismodel accounts for the 20- to 50-fold in-ducibility of GCN4 translation.
Genetic evidence indicates that reinitiat-ing ribosomes bypass uORFs 2 to 4 underderepressing conditions because the distance
between uORF1 and uORF4 is not largeenough to insure that they rebind the req-uisite factors required for reinitiation beforereaching uORF4. The key evidence for thisconclusion was that ribosomes were preventedfrom reaching GCN4 by gradually increasingthe separation between uORFs 1 and 4 byinserting spacer sequences. When theuORF1-uORF4 separation is expanded to
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Exogenous amino acid levels high
charged tRNA levels high
Translation rate high
GCN2 non-ribosome bound, inactive
eIF2α unphosphorylated
Ternary complex levels high
Amino acid levels reduced
uncharged tRNA levels high
GCN2 ribosome bound, active
eIF2α phosphorylated
Ternary complex levels reduced
Bulk translation decreases
GCN4 mRNA translated
Gcn4p dependent transcription of amino acid biosynthesis genes occurs
The GAAC response a translation control pathway that allows for survival when times are tough
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1] Global regulation
2] Message specific regulation
Two Types of Translational Control