Control of Eukaryotic Translation

8/16/2019 Control of Eukaryotic Translation

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Control of eukaryotic

translation

David Fear

[email protected]

DNA ProteinRNA

Text book molecular biology

Reverse transcription(retroviruses e.g. HIV)

Transcription Translation

Replication

DNA ProteinRNA

Points of regulation

Reverse transcription

(retroviruses e.g. HIV)

Transcription Translation

Replication

Regulation of cell cycle

Activation or repression

Stability of mRNAInitiation of translation

Blocking ofelongation

Post translational modification

(phosporylation, glycosylationProteolytic cleavage,

Degredation)

•

Understand the molecular mechanisms underlying

translation initiation, elongation and termination.

•

Be familiar with the “key” subunits and factors that areinvolved in the 3 phases described above.

•

Be able to describe specific examples where each of

these three phases is regulated to exert control over

protein translation.

ProteinRNA

Translation

Learning outcomes



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• Translation is mediated by the ribosome.

• Large complex of proteins and RNAs

(ribonucleoprotein)

•

Small subunit – “reads RNA”.

• Large subunit – joins amino acids onto

polypeptide chain.

Raza & Galili Nat. Rev. Cancer 2012

Ribosome Translation of mRNA by the ribosome

•

Ribosome moves from the 5’ to the 3’ end of the mRNA

• Amino acids delivered on tRNA, “reading” the triplet codon

• Amino Acids added to carboxyl end of polypeptide (C-term)

5’

mRNA 3’

Structure of a typical mRNA

5’ Methylated GTP ‘Cap’

• Regulates nuclear export of mRNA

• mRNA stability• Critical for translation initiation

• binds eIF4E

Internal ribosome entry site – IRES

• Allows translation independently of Cap recognition

Poly A tail

• ~100-200 Adenosines addedto 3’ end

• Effects mRNA stability

5’ untranslated region (5’UTR) 3’ UTR

UTRs – Untranslated regions

• Control mRNA stability & translation

efficiency (miRNAs)• May contain IREs – iron metabolism

Protein translation consists of 3

steps

• Initiation –

Complex event involving < 25 proteins

– Highly regulated

– Rate limiting

• Elongation –

Rapid, less regulation

• Termination – Dissociation of ribosomal complex when

stop codon (UGA) is encountered



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1. Initiation 2. Elongation

Protein translation consists of 3

steps

3. Termination

1. 40S ribosomal subunit associates with eIFs (43S complex)

2. 43S complex is targeted to the 5’ end of mRNA (48S complex)

3. Once bound, complex scans mRNA for AUG “start” codon

4. 60S subunit associates, Met-tRNA attached, translation begins

1: Translation initiation

Small ribosomal subunit, eukaryotic initiationfactors (eIFs), initiating tRNA (Met-tRNA) and

mRNA form the initiation complex

Involves the sequential binding of the small 40Sribosomal subunit first.

The large 60S ribosomal subunit binds once the start

codon is found.

40S

60S

80S


But re-association of the 40S/60S subunits is favoured

40S

60S

eIF1AeIF3

- eIF3 & eIF1A bind to the newly released 40s subunits,

- Prevents re-association with the 60S sub unit

80S

1.1: Formation of 43S pre-initiation complex




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40S

eIF1A

eIF3

eIF2 eIF2 binds and bringsinitiator tRNA (Met-tRNA)

With it

Met-tRNA

1.1: Formation of 43S pre-initiation complex


eIF4F (A/E/G)eIF5 eIF1A

eIF2

eIF3

40S

1.2: 43S complex attaches to the mRNA

AAAAAAAAAA

eIF4F (EIF4E) binds to 5’ methyl guanosine cap of mRNA

eIF4F40S

5’ cap 48S

complex

Met-tRNA

43S pre-initiation complex is formed when eIF4F & eIF5 bind

43S complex


1.3: Complex scans for AUG start codon (EIF4A is helicase)

AAAAAAAAAA eIF4F

40S

AAAAAAAAAA eIF4F

40S

AUG

AUG


5’ cap

5’ cap

AAAAAAAAAA

40S

1.4: once AUG is found, the complex will stop scanning. The60S ribosomal sub-unit associates & all initiation factors

dissociate.

AAAAAAAAAA

80S

60S

eIFS

ELONGATION

AUG




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Regulation of initiation

eIF1A

eIF2eIF3

40S

two major sites at which regulatory mechanisms act

1. eIF4F

2. eIF2

eIF4GeIF4AeIF4EeIF4F

complex

Regulation of initiation by eIF4F

1. eIF4F recruits mRNA to the ribosome via the 5’ cap.2. Levels of eIF4E (component of eIF4F) are low relative to

other initiation factors: potentially rate limiting.3. Phosphorylation of eIF4E seen when dormant cells are

stimulated with growth factors – promotes initiation.

4. Overexpression of eIF4E in mouse cells causes malignanttransformation.

5. Elevated elF4E is highly associated with cancer and is a

target for anti cancer drugs.

6. elF4E binding proteins (eIF4E-BP): Bind to eIF4E and

prevent initiation complex formation.phosphorylation of eIF4E-BP by growth factors prevents it

from binding eIF4E - activates translation.

eIF4E binds the 5’ cap of the mRNA

BUT many mRNAs are polyadenylated at the 3’ end

eIF4G binds poly A binding protein (PABP)

40S

AAAAAAAAAA 4E 4G

PAB

P

eIF4F complex binds mRNA

5’ cap

PABP binds the poly A tail

this interaction circularizes the mRNA

40S4E 4G

PA

B

PAAAAAAAAAA

Circularization enhances translation by allowing ribosome re-initiation by transfer of 40S ribosome from 3’ end to 5’ end,

without dissociation from mRNA.

eIF4F complex binds mRNA

5’ cap



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eIF4F

eIF1A

eIF2

eIF3

40S

Regulation of initiation

two major sites at which regulatory mechanisms act

1. eIF4F

2. eIF2

eIF2 provides the energy for the association

of the 60s ribosome subunit by GTP

hydrolysis

Regulation of initiation by eIF2

Integrated stress response:Viral infection (dsRNA),

metabolite starvation, ER

stress, Hypoxia and Haem

deficiency activate specific

Kinases.

• eIF2 is composed of three subunits eIF2 !, ", #.

• eIF2! binds GTP – hydrolysed to GDP!Energy

•

eIF2" exchanges GDP for GTP – for next round of translation• eIF2! can be phosphorylated at ser 51 by a Kinases• eIF2! phosphorylation inhibits eIF2" – Inhibits initiation

2: Elongation

t-RNAs deliver Amino acids to theribosome and “de-code” the codons.

The ribosome has 3 t-RNA binding sites:

A site – accepts incoming t-RNA and

forms peptide bond withgrowing peptide chain

Held in P site.

P site – holds t-RNA andthe growing peptide chain

E site – holds t-RNA without

Its amino acid.

tRNAs

tRNA “decode” the codon sequence of themRNA structure and deliver the amino

acid to the ribosome.

tRNA is folded into compact “cloverleaf “

structure with Aa attached to the 3’ end.

tRNAs are delivered to the ribosome

attached to elongation factors eEF-1,

helps to decode codon and provides

energy.



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Wobble hypothesis

The mRNA codon consists of threebases, recognised by the

complementary bases of the anti-codonon the tRNA.

BUT the 3’ position of the codon allowsnon-Watson-Crick base pairing –

“wobble” in the decoding.

Reduces the number of tRNA species

needed to decode the codons.

2: Elongation

The energy required to add the amino acid and move theribosome is provided by the hydrolysis of GTP at several

steps in reaction

Steitz Nat rev. Mol. Cell Biol. 2008

Elongation may be stalled due to miRNA (regulatory RNA)

binding to the mRNA by sequence

homology, often in the 5’ UTR.

miRNA is bound to RISC(RNA induced silencing complex)

which inhibits elongation

and can also induce mRNAdegradation).

Nelson P. Trends Biochem. Sci 2003

2: Elongation

Regulation of elongation:

Phosphorylation of elongation factor eEF2 inhibitselongation.

eEF2 can be phosphorylated by MAP kinase cascade

(growth factors), or in response to starvation, hypoxia and

stress via Protein Kinase A signaling or via the cell cycleregulatory proteins.

2: Elongation

Regulation of elongation:



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3: Termination

• Occurs when one of the stop codons appears (UAA,UAG, UGA)

•

No t-RNAs correspond to these codons so thepolypeptide chain is not extended.

• Ribosome release factor (eRF1) binds when stop codon

is in A site.•

eRF3 is a GTPase and is required to stimulate

polypeptide release.

• The 80s ribosome is now unstable and breaks apart.

Regulation of translation

1. Growth factors / Hormones

2. Cell Cycle

3. Viral infection

4. Structures within mRNA 5’UTR regulate translation

IRES (internal ribosome entry sites)

IRE (iron responsive element)



2. Cell Cycle

3. Viral infection




Ras

1. Growth factors and Hormones can up regulateTranslation via eIF4E phosphorylation

Insulin receptor

e.g Insulin

RafP

ERKP

MEKP

Cell proliferation

Binds cap more efficiently -

Up-regulates translation

eIF4EP

Mnk1


Ligand binding initiates theMAP kinase cascade,

resulting in activation of Mnk1which phosporylates eIF4E



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PI3K

1. Growth factors and Hormones can up regulateTranslation via eIF4E-BP phosphorylation

Insulin receptor

e.g Insulin

PDK1

mTORP

AKTP

Cell proliferation

Prevents eIF4E sequestration –

Up-regulates translation

eIF4E-BPP


Ligand binding initiates theMAP kinase cascade,

resulting in activation ofmTOR which phosporylates

eIF4E-BP

PIP2

PIP3

1. Stress response (Viral infection, metabolite starvation, ERstress etc.) activates specific Kinases that phosphorylate eIF2!

– Inhibits initiation.




2. Cell Cycle

3. Viral infection

4. Structures within mRNA 5’UTR regulate translationIRES (internal ribosome entry sites)


2. Cell Cycle

Translation is blocked at G2/M:

Employs mechanism previously discussed

•

eIF4E phosphorylation reduced – inhibiting initiation

•

Increased phosphorylation of eIF2 inhibits initiation

• Increased phosphorylation of eEF2 – inhibits elongation• Specific genes with IRES sequences still translated – see next

• Eukaryotic cell cycle •

S Phase - chromosome

replication

• M Phase - chromosome

segregation

•

G1, G2 gap phases




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2. Cell Cycle

3. Viral infection




- Virus infection induces Stress response: Inhibits Intitiation

and Elongation.- In addition, Picornaviruses: e.g. poliovirus &

Encephalomyocarditis virus (EMCV) specifically inhibit

translation of host mRNA by cleavage of translation factors

elF4G and PABP

!but viral mRNA is efficiently translated.

AAAAAA

AAAAAA

4G

3. Viral infection


5’Cap

5’Cap

Initiation of viral mRNA translation

is cap independent

All Picornavirus mRNA have multiple AUG codons, up to1.3kb away from 5’ end and internal ribosome entry sites

(IRES elements).

Therefore recruitment ofribosomes to viral mRNA

does not need a 5’ cap and

escapes inhibition bymechanisms preventing

initiation.



2. Cell Cycle

3. Viral infection

4. Structures within mRNA 5’UTR regulate translationIRES (internal ribosome entry sites)




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Scheper et al Nat Rev 2007

•

Common in virus mRNAs.

• Also present in mRNAs required during stress response,

mitosis and apoptosis.

• Allow translation of these mRNAs when cap dependent

initiation is blocked

Internal ribosome

entry site (IRES)forms stem loop

structures.

Control of translation by IRES Steric hindrance of translation via

IRE-IRP interactions

Gebauer & Hentze Nat Rev 2004

• Iron response elements (IRE) are present in mRNAs for transferrin

receptors and ferritin (iron storage metabolism).

• IRE-binding proteins (IRP) bind IRE and block translation of ferritin(iron storage) BUT promotes translation of transferrin receptors (iron

transport into cell).

• When iron concentration is high it binds IRP, prevents IRE binding,allowing translation of ferritin BUT decreasing transferrin receptors.

• Increases Iron storage and reduces iron transport into the cell.

eIF4E

eIF1A

eIF2

eIF3

40S

eIF4G

Summary – regulation of translation

eIF4E: phosphorylation STIMULATES translation

eIF4G: binds poly A tail via PABP STIMULATES translation

eIF2: phosphorylation of ! sub-unit INHIBITS translationeEF2: phosphorylation INHIBITS translation

UTRs: Bind miRNAs/RISC – INHIBITS translation

IRES: ALLOW translation when initiation is blocked

IRE: Control Iron metabolism

Jackson, R.J., Hellen, C.U.T. & Pestova, T.V. (2010) The mechanism

of eukaryotic translation initiation and principles of its regulation. NatRev Mol Cell Biol, 11, 113-127.

http://dx.doi.org/10.1038/nrm2838

Kleijn, M. et al., (1998) Regulation of translation initiation factors bysignal transduction. European journal of Biochemistry 253,

531-544

Sachs, A.B. et al., (1997) Starting at the beginning, middle and end:translation initiation in eukaryotes. Cell 89, 831-838

Proud, C.G. et al., (1997) Molecular mechanisms for the control of

translation by insulin. Biochemical Journal 328, 329-341

Pyronnet, S. & Sonenberg, N. (2001) Cell-cycle-dependenttranslational control Current opinion in genetics and development

11, 13-18

Further reading

Documents

Control of Eukaryotic Translation