Molecular Determinants of Accurate Translation Initiation

How do ribosomes identify the correct translation initiation codons in mRNAs?

Hinnebusch Lab (NICHD)

Lorsch Lab (NIGMS/NICHD)

Ramakrishnan Lab (MRC, U.K.)

Translation initiation by the scanning mechanism

AUG

scanning

…

GTP

40S

Met-tRNAi

5’ capmRNA

AUG

5’ cap

eIF4F

43S

…

GTP

TCGTP

eIF2

Met-tRNAi

eIF1

GTP

eIF1A

AUG…

GTP

mRNA

40S

scanning

43S PIC assembly

PIC attachment to mRNA

AUG

5’ cap

eIF4F

43S

…

TCGTP

eIF2

Met-tRNAi

AUG…

GTP

GTP

eIF1

GTP

eIF1A

mRNA

40S

scanning

GDP

Pi

AUGrecognition

…

eIF1

60S

80S IC

Protein synthesis

48S

eIFs

Scanning favors initiation at 5’-proximal AUGs

AAAAUGG-3 +4

AAAAUGG

GDP

Pi

eIF1

AAAAUGG-3 +4

5’

scanning

AAAAUGG-3 +4

UUUAUGU-3 +4 AAAAUGG

-3 +4

Poorcontext

Optimalcontext

UUUAUGU-3 +4 AAAAUGG

GDP

Pi

eIF1

“leaky” scanning

…and near-cognate triplets in good context can be used instead

AAAUUGG-3 +4

AAAAUGG

GDP

Pi

eIF1

AAAAUGG-3 +4

5’

scanning

AAAAUGG-3 +4

Near-cognate

Translation initiation defects in human disease

• Mutations adding or removing upstream AUGs or changing AUG context: melanoma, breast cancer, thalassemia, thrombocytemia, hereditary pancreatitis, familial hypercholesterolemia

• Overexpression of eIFs: malignant transformation.

• Mutations affecting eIF2B, the GEF for eIF2: leukoencephalopathy with vanishing white matter.

• eIF2g mutation: intellectual disability

• eIF1A mutations: uveal melanoma (UM) and thyroid carcinomas

NIF1A

eIF1SE

40S

“Open”

e

eIF1 and eIF1A promote “open” conformation of the 40S

eIF1 and eIF1A promote “open” conformation of the 40Sconducive to TC loading and scanning…

N UUG

“POUT”

GTP

“Open”, Scanning

NeIF1A

eIF1SE

40S

“Open”

GTP

TC

eIF1 and eIF1A promote “open” conformation of the 40Sconducive to TC loading and scanning…

NeIF1A

eIF1SE

N UUG

GTP GDPPi Pi

40S

“Open” “Open”, Scanning

GTP

TC

“gatekeeper”

“POUT”

NeIF1A

eIF1SE

N UUG

GTP GDPPi Pi

AUG

“PIN”

SEeIF1

GDP

Pi

40S

“Closed”, Initiation“Open” “Open”, Scanning

GTP

TC

“POUT”

…but eIF1 must be ejected to allow Pi release and stabilize TC binding in PIN state

AUG

“Closed”, Initiation

SE

eIF1

NeIF1A

eIF1

“Open”

SEN UUG

“Open”, Scanning

GTP GDPPi Pi

eIF1

GDP

PiGTP

TC

“POUT”

“PIN”

eIF1 promotes POUT for scanningand blocks PIN at non-AUG codons…

…requiring eIF1 release for AUG selection

N UUG

“Open”

NeIF1A

eIF1*

SESI

* *

GTPTC

“POUT”

Prediction: eIF1 mutations that weaken 40S binding shouldreduce TC binding to open complex in POUT state…

UUG

*

“Closed”

N UUGNeIF1A

eIF1*

SESI

* *

SE

GTPTC

“Open”

“PIN”

“POUT”

Prediction: eIF1 mutations that weaken 40S binding shouldreduce TC binding to open complex in POUT state…

…but allow transition to PIN at UUG codons

Translational Control of GCN4 by phosphorylation of eIF2

AUG AUG AUG

Translational Control of GCN4 by phosphorylation of eIF2

AUG AUG AUG

Integrated Stress Response by phosphorylation of eIF2

GCN4 translation: in vivo reporter of defective TC loading on 40S subunits

OFF

Rapid binding

Repressed

TC

GTP

ON

Slow bindingeIF* mutant

TC

GTP

Amino acid biosynthetic

genes

TC 40S: GCN4-lacZ expression

kON

(Gcd-)

GCN4 translation: in vivo reporter of defective TC loading on 40S subunits

Time

32P-TC

48S

PIC

Kinetics of TC loading

TC

GTP

40S

AUG

GTP

*

*Gcd- mutations:•eIF1 (sui1)•eIF1A (tif11)•18S rRNA•tRNAi

OFF

Rapid binding

Repressed

TC

GTP

ON

Slow bindingeIF* mutant

TC

GTP

Amino acid biosynthetic

genes

TC 40S: GCN4-lacZ expression

kON

(Gcd-)

Lorsch et al

AUG UUG

ACG UUG

ACG UUG

ACG UUG

HIS4

his4-301

his4-301 sui-

His+

His-

His+ , Sui-

His- , Ssu-

his4-301 sui- ssu-

-His UUG:AUG

low

low

increased

reduced

Sui- and Ssu- mutations alter accuracy of start codon selection

AUG UUG

ACG UUG

ACG UUG

ACG UUG

HIS4

his4-301

his4-301 sui-

His+

His-

His+ , Sui-

His- , Ssu-

-His

his4-301 sui- ssu-

UUG:AUG

low

low

reduced

increased

Sui- and Ssu- mutations alter accuracy of start codon selection

Quantify UUG/AUG initiation ratio:UUGHIS4-lacZ

AUGHIS4-lacZ

Prediction: eIF1 mutations that weaken 40S binding should reduce TC loading rate (Gcd- phenotype)…

UUG

*

“Closed”

Sui-

N UUG

“Open”

NeIF1A

eIF1*

SESI

* *

“Closed”

GDP Pi

SE

GTPTC

40S

…and elevate UUG initiation (Sui- phenotype)

Gcd-

“PIN”

“POUT”

K60E

18SrRNA

K37E

eIF1

Rabl et al (Ban N.) Science 2011

40S-eIF1 crystal structure

UUGGUUG

*

GDP Pi

K60E

Pilar Martin-Marcos

“PIN”

eIF1 affinity for 40S dictates TC loading and initiation accuracy

K60E

18SrRNA

K37E

eIF1

0

0.2

0.4

0.6

0.8

1

0 100 200 300 400 500

WTK60EK60E/D61GK60E/Q84H

Frac

tion

of e

IF1

boun

d

[40S] nM

140S

1A1 40S

1A

K60E

WT

UUGGUUG

*

GDP Pi

K60E

Sui-: UUG/AUG initiation

Pilar Martin-Marcos & Jagpreet Nanda

Gcd-: GCN4-lacZ : TC loading

“PIN”

eIF1 affinity for 40S dictates TC loading and initiation accuracy

40S-eIF1 crystal structure

K60E

WTeIF1

140S

1A1 40S

1A

K60E,Q84H

UUGGUUG

*

GDP Pi

S

Gc

K60E

“PIN”

eIF1 Ssu-

ui-: UUG initiation

d-: TC loading

N UUG

GDP Pi

* K60E,D61G

K60E,Q84H

TC loading

UUG initiation“POUT”

0

0.2

0.4

0.6

0.8

1

0 100 200 300 400 500

WTK60EK60E/D61GK60E/Q84H

Frac

tion

of e

IF1

boun

d

[40S] nM

D61G Q84H

K60E

K60E,D61G

eIF1 affinity for 40S dictates TC loading and initiation accuracy

eIF1 affinity for 40S subunit is finely tuned foroptimum initiation accuracy

K30(K37) H77

(Q84) W54(D61)

K53(K60)

Sui-

eIF1 40S: UUG:AUG eIF1 40S: UUG:AUG

Ssu-

AUG

PIN

“Closed”

SE

eIF1

N UUG

“Open”

POUT

GTP GDPPi Pi

eIF1

GDP

Pi

“PIN”

“POUT”

eIF1 blocks transition to PIN at non-AUG codons…

48S PIC

PIN

Cryo-EM structures of yeast PICs at 4.0 Å

Hussain & Llacer et al (Ramakrishnan)

• Assembled using Sui-

mutants of tRNAi and eIF2characterized at NIH

48S PIC

40SeIF1eIF1A

Cryo-EM structures of yeast PICs at 4.0 Å

• Assembled using Sui-

mutants of tRNAi and eIF2characterized at NIH

Hussain & Llacer et al (Ramakrishnan)

Transition to PIN alters eIF1 location to alleviate clash with tRNAi

•

mRNA

tRNAi (PIN)

tRNAi (POUT): Hashem et al. (Frank)

eIF1 in 40SeIF1eIF1AeIF1 in 48S PIC (PIN)

likely facilitates eIF1’s dissociation for AUG selection

mRNA

•

Transition to PIN alters eIF1 location to alleviate clash with tRNAi

tRNAi (PIN)

tRNAi (POUT): Hashem et al. (Frank)

eIF1 in 40SeIF1eIF1AeIF1 in 48S PIC (PIN)

Anil Thakur: mutations in eIF1 loops that should diminish the clash stabilize PIN at UUG codons (Sui-)

N UUG

eIF1

GTP

eIF5

eIF1A

AUG

SE

GDP

Pi

NTTCTT

SI “toggling factor”

eIF1A

ScanningEnhancer

(SE)

ScanningInhibitor

(SI)“Open”Scanning

“Closed”Arrested

“PIN”“POUT”

Tails of eIF1A regulate transition from open to closed conformation

NTTCTT

eIF1A

XXScanningEnhancer

(SE)

ScanningInhibitor

(SI)

F G F E S D E F E F G N ASE2SE1

A A A A A A A A AA A AA

AUG

SE

GDP

Pi

Sui-: UUG initiation

Gcd-: TC loading

N UUG

eIF1

GTP

eIF5

eIF1A

“POUT”“PIN”

Saini et al Genes Dev

Mutating SE elements in eIF1A CTT decreases accuracy and impairs TC loading

NTTCTT

eIF1A

XX

X

ScanningEnhancer

(SE)

ScanningInhibitor

(SI)

F G F E S D E F E F G N ASE2SE1

A A A A A A A A AA A AA

17-21 Ssu-: TC loading

UUG initiation

Sui-: UUG initiation

Gcd-: TC loading

AUG

SE

GDP

Pi

N UUG

eIF1

GTP

eIF5

eIF1A

“PIN”“POUT”

Saini et al Genes Dev

Mutating SI elements in eIF1A NTT restores accuracy and rapid TC loading

N UUG

eIF1

GTP

eIF5

eIF1A

AUG

SE

GDP

Pi

NTTCTT

SI

eIF1A

ScanningEnhancer

(SE)

ScanningInhibitor

(SI)

“PIN”“POUT”

eIF1A NTT promotes the PIN state

eIF1A NTT interacts with AUG-anticodon helix

Hussain & Llacer et al (Ramakrishnan)

• Ssu- mutations in the eIF1A NTT impede startcodon recognition

eIF1A NTT interacts with AUG-anticodon helix

Conserved bases in tRNAi play distinct roles in the accuracy of AUG selection

Met

tRNAi

AGCGCGGU

GGCGCAGUGGA AG C G C G

CAGGG

CU

C A UAA

CC

CUG A U

GUC

C U C GG A UCGAAACCGAGC

GGCGCUACCA

29 41

1 72

Anticodon

G31AG31CG31U Ssu-

G70AG70CG70U

U U

ASL

T loop

Sui-, Gcd-

17-21

eIF1A

* SI

C3

G70

Met

tRNAi

Jinsheng Dong

Disruption of C3-G70 confers Sui- and Gcd- phenotypesco-suppressed by eIF1A NTT mutation

G70A mutation decreases rate of TC binding in vitro…

N UUG

POUT

GTP

kON: 25X

Gcd-

GTP

TC

G70A

*

*

*

G

AGCGCC

C

ACCAUCCGG

Acc-stemG A

Time

32P-TC

48S

PIC

Kinetics of TC loading

40S

G70A

Tony Munoz (Lorsch lab)

N UUG

POUT

GTP

kON: 25X

Gcd-

GTP

TC

G70A

*

*

*

17-21

eIF1A

*

17-21

*

60XG

AGCGCC

C

ACCAUCCGG

Acc-stemG A

G70A

…in a manner reversed by eIF1A NTT mutation 17-21

Tony Munoz (Lorsch lab)

Base-pair substitutions of G31-C39 confer Sui- but not Gcd-

phenotypes

AGCGCGGU

GGCGCAGUGGA AG C G C G

CAGGG

CU

C A UAA

CC

CUG A U

GUC

C U C GG A UCGAAACCGAGC

GGCGCUACCA

29 41

1 72

Anticodon

A UU AC G

Sui-G31AG31CG31U Ssu-

G70AG70CG70U

U U

Sui-, Gcd-

ASL

C3

G70

A54

A60

G29

C41

G30G31

ASLG:Cpairs

C40C39

tRNAi

Met

Jinsheng Dong

UUGN UUG

Sui-

POUT PIN

Stabilized

GTP GDP Pi

**

C3

G70

A54

A60

G29

C41

G30G31

C40C39

G70A

U31:A39

Met

ASLG:Cpairs

*

Hypothesis: U31:A39 substitution in ASL removes barrier to PIN

koff: ~high koff: low

AUG

WT <0.4 1.2

AUG UUG

N UUG

TC is less tightly bound to the PIC at UUG codons

koff: 1.2/hr

N UUG

WT

UUG

koff:<0.01/hr

Sui-

*

CAGGG

CU

C A UAA

CC

CUG

29 41

U A

ASL

U31:A39

WT

mRNA(UUG)

U31:A39 (Sui-)

C31:G39 (Sui-)

U31:A39 replacement stabilizes PIN at UUG codons

Tony Munoz (Lorsch lab)

G31:C39 impedes PIN state & demands perfect AUG-anticodon duplex

AUGN UUG

POUT PIN

SE

GDP Pi

G31:C39

C3

G70

A54

A60

G29

C41

G30G31

ASLG:Cpairs

C40C39

GTP

…

Hussain & Llacer et al (Ramakrishnan)

PIN

POUT

tRNAi anticodon stem is distorted in PIN state

N AUC

eIF1

GTP

eIF5

eIF1A

SI

“Open”Scanning

“POUT”

AUG

SE

GDP

Pi

“Closed”Arrested

“PIN”

Evidence for 40S conformational changes was lacking

N AUC

POUT

eIF1

GTP

eIF5

eIF1A

AUG

PIN

SE

GDP

Pi

NTTCTT

SI

eIF1A

“Closed”Arrested

Cys::Fe(II)BABE

Cys::Fe(II)BABE

Cys::Fe(II)BABE

“PIN”“POUT”

Structural probing of PICs by free-radical cleavage directed by eIF1A

Fan Zhang & Adesh Saini

A1446C1447G1448

C1461G1462C1463

N105CC1210A1211G1212G1213

40S

body

head

tRNAi

mRNA

eIF1A

Structural probing of PICs by free-radical cleavage

directed by eIF1A

Fan Zhang & Adesh Saini

Greater cleavage of P-site residues in “open” (AUC) versus “closed” (AUG) complex

40S

onl

y

U G C AC

ys-le

ssK

11C

P22

CV

69C

Q73

CV

129C

V12

9C/S

E*

D13

7C

K11

C/S

E*

G14

3C

No mRNA

Cys

-less

K11

C

P22

CV

69C

Q73

CV

129C

D13

7CG

143C

Cys

-less

K11

C

P22

CV

69C

Q73

CV

129C

D13

7CG

143C

AUG AUC

V12

9C/S

E*

K11

C/S

E*

V12

9C/S

E*

K11

C/S

E*

U1578

G1575A1577A1576

eIF1A

Cys::Fe(II)BABE

Cys::Fe(II)BABE

Cys::Fe(II)BABE

Open/POUTClosed/PIN

Fan Zhang & Adesh Saini

AUG recognition evokes closure of P site (PIN)

A1576

A1577U1578G1462

C1463

C1634A1635

C1637G1638

A1756G1757

C1759G1760

U1761

U1643-C1646

C1439G1440 A1425-

G1429

U558C559

G574C575G552

G553

C1461

U1758

G1575

A579

mRNA

Cleavages in P-site and mRNA binding cleft suppressed in AUG vs AUC complex

• AUG recognition constricts mRNA binding cleftand closes the P site

Open PIC conformation at AUC shows upward movement of 40S head

Llacer et al (Ramakrishnan)

•

py48S-open: (AUC)mRNA

py48S-closed: (AUG)mRNA

Conducive for mRNA recruitment & scanning

AUC

AUC AUG

Open PIC conformation at AUC shows widened P-site

• Compatible with triplet sampling by tRNAi during scanning

Llacer et al (Ramakrishnan)

eIF2β contacts tRNAi, eIF1, and eIF1A inopen complex

py48S-open

Llacer et al (Ramakrishnan)

eIF2β contacts eIF1 exclusively in open complex

Closed (AUG) initiation

eIF1

tRNAi

eIF2β

Ser202

Lys214

Phe217

Gln221 mRNAα4

α3α2

open (AUC): scanning

eIF1

tRNAi

Ser202

Lys214

mRNA

α4eIF2β

α3

α2

eIF2β mutations eIF1 mutations eIF2β mutations

Laura MarlerAnil Thakur

eIF1

tRNAi

eIF2β

Ser202

Lys214

Phe217

Gln221 mRNAα4

α3α2

open (AUC): scanning Closed (AUG) initiation

eIF1

tRNAi

Ser202

Lys214

mRNA

α4eIF2β

α3

α2 mutations

eIF2β contacts with eIF1 promote scanning and impedeUUG initiation

mRNA

head

body

mRNA

eIF2α

z

R225

E144

R156

R148

R157

-3A

UG

Rps5tRNAi

mRNA

Jyothsna VisweswaraiahYvette Pittman (Dever lab)

Rps5 hairpin substitutions suppress UUG initiation

eIF2α

z

R225

E144

R156

R148

R157

GCN4-lacZ

AUG

el. uORF1

AUG

rps5-E144R

GCN4-lacZ

AUG

el. uORF1

AUG

WT

-3A

UG

Rps5tRNAi

mRNA

Jyothsna VisweswaraiahYvette Pittman (Dever lab)

rps5-E144R impairs AUG recognition by the scanning PIC

1st AUG blocks initiation downstream

Bypass of 1st AUG enables initiation

downstream

koff

AUG

40X

GCN4-lacZ

AUG

el. uORF1

AUG

rps5-E144R

Bypass of 1st AUG enables initiation

downstream

GCN4-lacZ

AUG

el. uORF1

AUG

WT

1st AUG blocks initiation downstream

E144R 40S

Jyothsna Visweswaraiah

•

rps5-E144R impairs AUG recognition by destabilizing PIN state

Rps5 is on par with eIFs in controlling AUG recognition

“Open”Scanning

“Closed”Arrested

Conformational rearrangements in transition from scanning to AUG selection

• Downward head movement constricts mRNA cleft• P site closes around tRNAi

• eIF1A NTT interacts with codon:anticodon duplex• eIF1 displaced by tRNAi from P site

• eIF1 dissociates to allow Pi release from eIF2

MRC Laboratory of Molecular Biology, University of Cambridge, UK

Not shown:Israel Fernandez

Venki Ramakrishnan

Tanweer HussainJose Llácer

Lorsch LabShardul Colin Paul Sarah Jagpreet

Jon

Not shown: Tony Munoz & Fujun Zhou

Funding: NIH

JyothsnaFan

Anil

Dong

QuiraHongfang

Laura

DavidYashpal Neelam

Neha

Alan’s Lab

Not shown: Suna Gulay, Pilar Martin-Marcos, Adesh Saini

Alan’s Lab

JyothsnaFan

Anil

Dong

QuiraHongfang

Laura

DavidYashpal Neelam

Neha

Not shown: Suna Gulay, Pilar Martin-Marcos, Adesh Saini

Tom Dever