The initiation phase of protein synthesis in eukaryotescase.edu/med/coller/Coller C3MB Lecture...

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



2] Message specific regulation


• 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


eIF1AeIF2 eIF2BeIF3eIF4AeIF4BeIF4EeIF4GeIF5eIF5Baminoacylated initiator methionyl tRNA (Met-tRNAi

Met)

40S ribosomal subunit60S ribosomal subunitGTP ATP


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


Ternary Complex-TC

3 2 GTPMet

1 1A

1

Formation of Ternary Complex an important regulatory node

Met-tRNAiMet


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


3

AUGCap4E 4G

4A4B


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


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

0 min

P

80S

RNP

120 min

GAL-Dhh1pCondition A Condition B


0 min

P

80S

RNP

120 min

GAL-Dhh1pCondition A Condition B


Usually seen during conditions of stress or stimulation by hormone

Ternary Complex formation

eIF-4E regulation


3 2 GTPMet

1 1AAUGCap

4E 4G4A4B

43S mRNA interactioneIF3 in the 40S complex and eIF4G in the mRNA complex interact


3 2 GTPMet

1 1A


3

AUGCap4E 4G

4A4B


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)

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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

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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


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.

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4E-BP proteins block eIF4E/eIF4G binding

eIF4G peptide

4E-BP peptide

Gingras et al., 1999


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

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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


60S

40S 40S3

60S

1A3 1A

2GDP

2 GTP

GTP

GDP

Met

2 GTPMet

2B


Ternary Complex-TC

3 2 GTPMet

1 1A

1


Met-tRNAiMet


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

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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


Ternary Complex-TC

3 2 GTPMet

1 1A

1


Met-tRNAiMet



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

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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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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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The initiation phase of protein synthesis in eukaryotescase.edu/med/coller/Coller C3MB Lecture...

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Transcript of The initiation phase of protein synthesis in eukaryotescase.edu/med/coller/Coller C3MB Lecture...