Translation II

Gerhard WagnerBCMP200 12/04/2002

wagner@hms.harvard.edu

2

Translation initiation - big picture

• In the absence of mRNA, ribosomes are dissociated

• Translation is initiated by recruitment of f-Met-tRNAMet and mRNA to the small ribosomal subunit

• Recruitment is facilitated by a number of initiation factors

• Initiation is complex in eukaryotes but simpler in prokaryotes

• There are many more levels of control in eukaryotes

3

Initiation prokaryotes vs. eukaryotes

prokaryotes eukaryotes

From: Hershey & Merrick in “Translational Control of Gene expression” pp. 33-88”, Sonenberg, Hershey, Matthews, eds. CSH Press 2000

4

Translation initiation in prokaryotes

• There are few Initiation factors– IF1 (7.1 kDa)

• binds after ribosome dissociation, • binds to A-site of small subunit • assists IF2 and IF3

– IF2 (79.7 kDa), homologous to eIF5B • binds fMet-tRNAi • GTPase

– IF3 (20.7 kDa) • ribosome anti-association• Involved in tRNAi-codon interaction, decoding• Assures together with IF2 that fMet-tRNAini is bound not any other

– W2 (71 kDa), ATPase, eIF4A (helicase) homolog?• Little known, no solid evidence for helicase function, stimulates in-vitro

translation of some mRNA with secondary structure around AUG – EF-P (21 kDa), eIF5A homolog

• Little known

5

Model for Initiation in Prokaryotes

• Dissociation of ribosome due to absence of mRNA and aided by IF3 binding to 30S

• IF3 was the earliest- and most-studied bacterial initiation factor

• Association to 30S of (which order is unknown)– IF1 into A-site– ternary complex (IF2, fMet-tRNAi, GTP) - places

fMet-tRNAi into P-site– mRNA at AUG codon (Shine Dalgarno) - decoding

task - get the AUG codon matching the anticodon of fMet-tRNAMet - GUG(8%) and UUG (1%) are sometimes used - internal AUGs vs initiation codons

• This is followed by dissociation of IF1 and IF3• 50S particle joins• IF2 is released with GTP hydrolysis

– (what is the GEF for IF2?)• Elongation starts• What is known structurally?

– IF1 (eIF1A) has OB-fold and has been located on the 30S particle

– IF2 (eIF5B) structure solved and approximate placement on 30S has been mapped with footprinting experiments

– IF3(no eukaryotic homolog) N- and C-terminal domains known and partial structures on 30S particle

6

Role of IF3 and initial placement fMet-tRNAf an 30S subunit

• IF3 was originally most studied initiation factor. Was identified as a dissociation factor, because it binds the 30S subunit with high affinity and thereby prevents the formation of 70S ribosome. This binding results in a shift in the equilibrium toward the dissociation of 70S ribosomes into 50S and 30S subunits.• Cryo-EM studies from the Frank laboratory provided a first structural picture for the location of IF3 (McCutcheon et al., PNAS 1999)• The crystal structure of IF3 alone had been determined before and was fitted to cryo-EM difference maps

Crystal structure of IF3fitted to cryo-EM densityOver-interpretation of shape?

7

Mapping initiation factor binding sites Mapping of initiation factor binding sites on 30 S subunit with

Footprinting Moazed et al. JMB 248, 207-210 (1995): IF1, IF3, IF2

CrosslinkingTargeted hydroxyl radical footprinting

Dallas & Noller, Molecular Cell 8, 855 (2001)Datwyler & Meares, TIBS, 25, 408 & 468 (2000).

X-ray crystallography (IF1)see below, Carter et al., 2001

NMR spectroscopy (IF1 - fMet-tRNA)Meunier et al. EMBO J. 19, 1918 (2000)

8

Location of initiation factor IF1 on 30S particle

• Clemons et al., solved structure of 30S particle, JMB 1999, Science, 2001

• Carter et al. (2001) solved the structure of IF1 bound to the 30S particle

• Fig. shows 30S particle with IF1 bound close to S12 protein

• Landmarks:– Head (H), Neck (N), Platform (P), Body

(Bo), Shoulder (Sh)• Notice difference in shape to low-

resolution cryo-EM (McCutcheon)• Helix 44 and S12 interact with initiation

factor IF1, which sits in the A-site, adjacent to S12 protein and helix 44

IF1

S12 H44

Fig. 2B Carter et al., 2001

9

Carter et al., Science 2001, Fig. 2 • IF1 (OB-fold - seen before in aa-tRNA synthase) binds 30S subunit between H44, H18

and S12 and causes a conformational change in H44• Function of IF1 is to make sure that fMet-tRNAMet is placed into P-site• IF1 is likely to interact with the IF2 / fMet-tRNAMet /GTP ternary complex

IF1

S12

H44

H18

A PE

10

Positioning of IF3Location of IF3 has been studied with numerous crosslinking, footprinting, cryo-EM and

crystallographic experiments. Some of the results are a little contradictory and controversial Most recent and plausible data of Dallas & Noller (2001) are shown here.

Structures of free N- and C-terminal domains of IF3 have been solved independently (see above)

11

Directed hydroxyl-radical cleavage

Datwyler & Meares, TIBS 25, 408 & 468

(S)-1-(p-bromoacetamidobenzyl) EDTA (FeBABE)

AscorbateH2O2

12

Positioning of IF3 relative to tRNAi and IF1 (Dallas & Noller, Mol. Cell (2001)

A. Model of tRNAMetf relative to IF3, IF1 in A-site and mRNA in P-siteB. Model for the interaction of the anticodon of fMet-tRNA with

mRNA

Notice change in shape of IF3

mRNA decoding

13

Structure of IF2 (archaeal homolog of eIF5B)

Roll-Mecack et al., Cell 103, 708 (2000)

IF2 (eIF5B) binds to the small ribosomal subunit

Approximate positioning on ribosome from footprinting data

Domain II of bacterial IF2 interacts with IF1

Promotes binding of fMet-tRNAfMet to small ribosomal subunit

GTP hydrolysis upon release from ribosome and subunit joining

14

Summary Prokaryotic Initiation

• There are plenty of functional and structural data– Ribosome - structure and active site– IF1 location - place holder -directs fMet-tRNAi to P-site– IF3: aids ribosome dissociation location adjacent to

tRNAi– IF2 location mapped - needed for 50S joining

• Order of assembly still unclear• Mechanism of 50S joining ?

15

Eukaryotic Initiation - big picture

1. Recruit Met-tRNA to small particle

2. Recruit mRNA to small particle

3. Scan to AUG codon

4. Bind large particle and release initiation factors

5. Elongation starts

1.

2.

3.

4.

5.

16

Three connected processes

• Build up of small ribosomal particle

• Build up and delivery of ternary complex (eIF2/fMet-tRNAi/GTP)

• Build up and joining mRNA/initiation factor complex

17

Eukaryotic Translation Initiation - details

• Dissociation of ribosome binding eIF1A and eIF3 to 40S

• Recruit ternary complex (fMet-tRNAi, eIF2, GTP) to 40S

• Recruit mRNA to 43S -> 46S particle

• 46 S scans mRNA to reach AUG

• Initiation factors leave and 80S joins

• Elongation begins

• Highly controlled process• Loss of control -> cancer

18

Eukaryotic Translation Initiation - Details

• Dissociation of ribosome binding of eIF1A (A-site) and eIF3 to 40S• eIF3 is a huge complex of 11 subunits (MW = 0.69 MDa) - not homologous to IF3• Possibly eIF5, eIF5B and eIF1 bind at this point as well• eIF1 seems to play a similar role as IF3-C

• Recruit ternary complex (fMet-tRNAi, eIF2, GTP) to 40S

• eIF2 is a complex of three subunits (, , ) of 123 kDa• GTP is loaded by eIF2B• Regulated by phosphorylation

• Recruit mRNA to 43S• mRNA is bound to the eIF4F complex (4E,4G,4A)

• Scan mRNA to reach AUG• Scanning requires a defined number of eIFs

• Initiation factors leave and 80S joins • eIF5 is the exchange GEF for eIF2 -> eIF2 release •Ribosome is GEF for eIF5B -> eIF5B release

•Elongation begins

19

eIF1A (IF1 homolog) has OB fold plus an additional domain

and long flexible tails

Battiste et al., Mol. Cell,5, 109 (2000)

20

eIF1A binds RNA

mRNA?tRNA?rRNA?

IF1 complex with ribosomeCarter et al. Science 291,498 (2001)

Battiste et al. Mol. Cell 2000

H44H18H1

S12

22

Delivery of fMet-tRNA via eIF2

• eIF2/GTP delivers fMet-tRNA to the 40S particle and places it into the P-site

• eIF2 is a trimeric complex () of 127 kDa

• Binds fMet-tRNA when GTP is bound• eIF2B is the Guanylate exchange factor

(GEF) for eIF2• eIF2B is a pentamer of 261 kDa, no

structure is known• Ternary complex formation is inhibited by

phosphorylation of eIF2 at Ser 51• The ternary complex binds the 40S

subunit to form the 40S preinitiation complex (43S initiation complex)

• eIF3 is needed for binding the ternary complex

• Ternary complex stays on the ribosome until AUG codon is reached (after scanning). At this point, hydrolysis of eIF2-bound GTP is catalyzed by eIF5, eIF2-GDP leaves and fMet-tRNA stays on the ribosome

23

More on eIF2

• eIF2 is a 127 kDa trimeric complex highly conserved in eukaryotes– eIF2 (36 kDa) has a crucial phosphorylation site at S51– eIF2 (39 kDa) function not clear– eIF2 (52 kDa) GTP-binding

• There is no structure yet of entire eIF2 – N-terminus of eIF2 has OB fold– crystal structure of N-terminal half has been solved (Jon Clardy lab, JBC

277, 17057 (2002)– Ser51 sits on a flexible loop

• Ser 51 phosphorylation leads to a conformational change that causes formation of a tight complex with eIF2B and initiation is inhibited

24

eIF2 consists of three subunits

-subunit

P-Ser51 315

-subunit

Lys blocks Zn finger motif14 80 124 281305333

-subunit

GTP binding motif48 134 190 472

eIF5tRNAi

Met

eIF2B

eIF2B

PKR, HRIGCN2, PERK

25

Significance of Ser 51 phosphorylation• Ser51 is phosphorylated by kinases, such as

– double-stranded RNA-activted protein kinase PKR– PERK = PKR-like ER kinase– Heme-regulatd eIF2 kinase (HRI = heme-regulated inhibvitor)– GCN2 in yeast

• Reversible phosphorylation provides the cells with a mechanism to regulate the the amount of mRNA rapidly and reversibly in response to a variety of stimuli– PKR: response to double-stranded RNA virus – PKR-induced eIF2 phosphorylation can cause apoptosis– Iron deficiency -> heme deficiency activates HRI ->Ser51 phosphorylation

and downregulation of translation in erythrocytes– PERK regulates translation in the ER in response to stress

26

eIF4Am7Gppp

AAAAAA

AUG

eIF4E

eIF4G

Recruitment of mRNA to the 43S ribosomal subunit

eIF3

40S

eIF2Met-tRNAi

eIF1A

eIF4F = eIG4G/eIF4E/eIF4AeIF4G is a scaffold protein that binds eIF4E, eIF4A, Pabp, Mnk-kinase, eIF3eIF4E is cap-binding proteineIF4A is RNA helicase

27

cap

4EBPeIF4G

Structure of eIF4EMatsuo et al. 1997

N

35

28

Residues 1-33 are unstructured in free eIF4E

What happens when eIF4G binds?

29

eIF4G1393-490 folds upon binding eIF4E

free bound to eIF4E

Hershey, McWirther, Gross, Wagner, Alber, Sachs, JBC 1999

30

eIF4E/eIF4G/cap complex

31

N

C

C

N

cap

eIF4E

eIF4G

SummaryeIF4E/eIF4G/cap complex

N-terminal segment of eIF4E folds upon eIF4G binding

eIF4G wraps around N-terminus of eIF4E

Binding induces mutual folding to form a 35 kDa complex

32

m7Gppp

AAAAAA

AUG

eIF4E

eIF4G

eIF3

40S

Recruitment of mRNA to the 43S ribosomal subunit

eIF4A

Formation of this complex is highly regulated

Gross et al. 2003 Marcotrigiano et al.2001

Middle domain is a HEAT domain

Burley et al. 2003

NTD-eIF4A

CTD-eIF4A

eIF4A is a DEAD-box protein

Marcotrigiano et al.1997Matsuo e al. 1997

33

Regulation of translation initiation by 4E-BP • eIF4E binding to eIF4G is

inhibited by 4E-BP

• eIF4G and 4E-BP share a consensus binding sequence YxxxxL

• Growth factors or hormones may cause hyperphosphorylation of 4EBP , dissociation and formation of preinitiation complex

• Translation initiated

34

4EBP is unstructured and exhibits only minor changes upon binding eIF4E

free complex

35

YXXXXL

36

37

Hierarchical phosphorylation of 4E-BP

38

Initiation via Internal Ribosome Entry Sites

Some mRNAs have 5’UTRs that act as direct binding sites for small ribosomal particle - in concert with the middle domain of eIF4G

First identified for viral RNAsHepatitis C virus (HCV)Classic swine fever virus (CSFV)Foot and mouth disease virus

Now several cellular proteins have been found to be translated via IRESs

eIF4G, FGF2, VEGF etc..

39

Preinitiation complex in translation initiation

eIF4G

Recruitment of 40S to mRNA via eIF3>eIF4G>eIF4E>m7G>mRNA

Recruitment of 40S to mRNAvia eIF3>eIF4G>Pabp>poly(A)>mRNA

Recruitment of 40S to mRNAvia eIF3>eIF4G>IRES

4B/4H

Alan Sachs et al.

40

Eukaryotic Translation Initiation - Summary

• Complex eukaryotic translation initiation is to provide more control over protein production

• Many forms of cancer have elevated levels of initiation factors