The Initiation of Translation in Pro- and Eukaryotic Cells

5’ cap:

After 20-30 nucleotides have been

synthesized, the 5’-end of the mRNA is capped
5’ to 5’ with a guanine nucleotide. Essential for
the ribosome to bind to the 5’ end of the mRNA.

Poly (A) tail:

50-250 adenine nucleotides are added to

3’ end of mRNA. Stabilizes the mRNA, and plays
an important role in transcription termination.

Eu- and Prokaryotic Ribosomes

Eukaryotic cytoplasm

Prokaryotes, Eukaryotic organelles

(mitochondria, chloroplasts)

E: Exit site for free tRNA P: peptidyl-tRNA A: aminoacyl-
tRNA

E, P and A Sites of Ribosomes

Initiation of Translation in Prokaryotes

50S

30S

50S

30S

RRF

IF-3

mRNA

(fMet-tRNA

Met

)

IF-2GTPfMet-tRNA

met

GTP

fMet

30S Initiation Complex

IF-1: 71aa, assists IF-2 binding

IF-2: 890aa, binds initiator
tRNA
and GTP

IF-3: 180aa, releases mRNA
and
tRNA from recycled 30S
subunit and aids (new)
mRNA binding

RRF: ribosome release factor

IF-2 = initiation factor 2

In complex with GTP, it brings
fMet-tRNA

Met

to the partial P

site on the small subunit.

Activates a GTPase activity in
the small subunit, which
allows dissociation of IF2, IF3,
and IF1.

mRNA

GTP

fMet

GTP

fMet

30S Initiation Complex

GTP

fMet

+ GDP +Pi

fMet

70S Initiation Complex

fMet

site

Elongation Phase of Translation

Simple process – involves only initiation factors (IFs) IF-1, IF-2
and IF-3
plus….. fMet-tRNA

Met

and mRNA

mRNA binds to small ribosomal subunit such that initiator AUG
is positioned in the precursor to the P site

In eubacteria, such as E. coli, the positioning of the initiator
AUG is mediated by base pairing between

the ribosome-binding site in the (5’) untranslated region of the
mRNA
and the 3’ end of the 16S rRNA

Initiation of Translation in Prokaryotes

Some translational initiation sequences recognized by E. coli ribosomes.

Shine-Dalgarno (ribosome binding) sequence: A nucleotide sequence (consensus = AGGAGG)
that is present in the (5') untranslated region(s) of prokaryotic mRNAs. This sequence serves as a
binding site for ribosomes.

No involvement of mRNA 5’ end

Shine – Dalgarno sequences +AUG initiation codons can occur
within 5’ non-translated regions, and, may also occur within
site(s) internal to the mRNA …….

Prokaryotic mRNAs may be polycistronic

The ability to bind ribosomes and initiate translation at sites
internal to the prokaryotic mRNA allows

• genes to be organised into operons,

• an operon to be transcribed into a single (polycistronic) mRNA,

• the expression of a number of genes (related functions) to be
controlled
by a single promoter (or single translational control
mechanism)

cistron 1

cistron 2

cistron 3

sites of ribosome ‘re-cycling’

Initiation of Translation in Prokaryotes

Initiation of translation
in Eukaryotes

It’s (much) more complex ..

Initiation of Translation in Eukaryotes

major differences to prokaryotic mRNA……

• eukaryotic mRNAs possess a different 5’ ‘cap’ structure

• eukaryotic mRNAs are polyadenylated

Bases around the initiating AUG influence the efficiency of initiation:
RNNNAUGG (‘Kozak consensus’ sequence)

Eukaryotic initiation factor eIF4 scans along mRNA from cap to find
initiator AUG

(initiating)
AUG

open reading frame

A(n)

Stop codon

5’ ‘cap’

me7’

Gppp

43S

‘Scanning’ Model of Eukaryotic Initiation of Translation

(initiating)
AUG

open reading frame

A(n)

Stop codon

5’ ‘cap’

me7’

Gppp

‘scans’

(initiating)
AUG

A(n)

Stop codon

5’ ‘cap’

me7’

Gppp

(initiating)
AUG

A(n)

Stop codon

5’ ‘cap’

me7’

Gppp

60S

(initiating)
AUG

A(n)

Stop codon

5’ ‘cap’

me7’

Gppp

Elongation Phase

AAAAAAAAAAAAAAAA

m7Gppp

Messenger RNA structure

5’NCR

AUG

3’NCR

stop

Open reading frame

‘Cap’

Exon / exon splice boundaries

Poly(A) tail

m7Gppp

Messenger RNA structure – 5’ NCR

AUG

RNA stem-loop structures

AAAAAAAAAAAAA

orf

Messenger RNA structure – 3’ NCR

stop

A / U rich Elements (AREs)

- binding sites for stabilising
/ destabilising proteins

mRNA localisation elements

(usually located in the 3’NCR)

- binding sites for proteins which bind to the cytoskeleton

- binding sites for proteins (located at specific cellular sites)
which anchor the mRNA in that location

Initiation of Translation

60S

40S

60S

40S

eIF6

eIF3

Ribosome anti-association factor

Binds to 40S subunit (stabilises
Met-tRNAi) and prevents
association with 60S subunit

Ternary complex formation

eIF2

GDP

eIF2

GDP

eIF2B

eIF2

GTP

Met

eIF2

GTP

Met

eIF3

eIF5

ternary complex

eIF2

GTP

Met

multifactor complex (MFC)

eIF1

guanine nucleotide
exchange factor (GEF)

GDP-bound eIF2 cannot bind Met-tRNA

Met

GTPase activating protein (GAP)

initiator methionyl tRNA

eIF3

eIF5

eIF1

eIF3

eIF5

eIF2

GTP

Met

eIF1

43S Complex Formation

eIF3

eIF5

eIF2

GTP

Met

eIF1

40S

eIF1A

eIF1A and eIF3 promote binding
of the multifactor complex to the
40S subunit

43S Complex

multifactor complex (MFC)

eIF1A

eIF4G

eIF4E

PABP

eIF4A

Binds poly(A) Tails

Binds 7meG ‘caps’

eIF4E : cap binding
eIF4A : bi-directional RNA helicase
MnkI : MAP kinase-interacting protein kinase-1 (phosphorylates eIF3)
PABP: poly(A) binding protein

Assembly of the Cap Binding Complex:
Eukaryotic Initiation Factor 4G (eIF4G)

eIF3

eIF5

eIF2

GTP

Met

eIF1

40S

eIF1A

eIF4A

Binds
eIF3

eIF4E

eIF4G

eIF4A

Recruitment of the 43S complex to the 5’ end of the mRNA

eIF4B

eIF3

eIF5

eIF2

GTP

Met

eIF1

40S

eIF1A

GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

• eIF3 : interacts with eIF4G to recruit the 43S complex

• eIF4B : RNA binding protein; stimulates (but not essential for) ribosome
binding to natural mRNA

PAB
P

AAAAAAAAA

Scanning of the 5’ UTR
and AUG recognition

eIF4E

eIF4G

eIF4A eIF4B

eIF3

eIF5

eIF2

GTP

Met

eIF1

40S

eIF1A

GpppGAUUCGAUACCAGGGAGCUUGGC

ACC

AUG

eIF4E

eIF4G

eIF4A

eIF4B

eIF3

eIF5

eIF2

GTP

Met

eIF1

40S

eIF1A

GpppGAUUCGAUACCAGGGAGCUUGGC

ACC

AUG

ATP

ADP + P

cap-binding complex recycled

eIF3

eIF5

eIF2

GTP

Met

eIF1

40S

eIF1A

GpppGAUUCGAUACCAGGGAGCUUGGCACC

AUG

Conformational change, GTP hydrolysis,
release of initiation factors,
and assembly of the eIF5B GTPase

eIF3

eIF5

eIF2

GDP

eIF1

Met

40S

eIF1A

GpppGAUUCGAUACCAGGGAGCUUGGCACC

AUG

eIF5B

GTP

GTP hydrolysis by eIF2 requires eIF5 and possibly
a conformational change triggered by the
Met-tRNA

Met

interaction with the 40S subunit

Met

40S

eIF1A

GpppGAUUCGAUACCAGGGAGCUUGGCACC

AUG

eIF5B

GTP

Assembly of the 80S ribosome

40S

eIF1A

eIF5B

GDP

60S

GTP hydrolysis by eIF5B serves as a final
checkpoint for correct 80S assembly

the 80S ribosome is now poised to elongate

60
S

GpppGAUUCGAUACCAGGGAGCUUGGCACC

AUG

Met

Initiation of Translation of Eukaryotic mRNAs

nothing similar to prokaryotic Shine-Dalgarno sequence

(ribosome binding

site; RBS)

5’ mRNA cap structure crucial

cap-binding protein complex recruits 43S complex

‘scanning’ of mRNA by 43S complex from 5’ cap structure to initiating
AUG

assembly of 80S ribosome ….. elongation

termination (proximal to site of initiation!!)

mRNAs translated as circular complexes

(essentially) no mechanism for internal initiation – eukaryotic mRNAs
are monocistronic

Factor Mol mass (kDa) and Function (based on biochemical studies in mammals)
subunit composition

eIF-1 14 Omission restricts binding of 40S subunit to 5’ end (entry
site)
on mRNA
eIF-1A 17 Catalytically promotes Met-tRNAi binding to 40S;
required for
strong binding of 40S subunit to mRNA

eIF-2

36 (), 38 (), 52 () GTP binding protein; escorts Met-tRNAi onto 40S

ribosomal
subunit

eIF-2B

81, 71, 58, 43, 34 Guanine nucleotide exchange factor (GEF): promotes

exchange
of GDP for GTP on eIF-2

eIF-3

110, 67, 42, 40, 36, 35 Binds to 40S subunit, stabilizing Met-tRNAi and

preventing
association with 60S subunit

eIF-4E

25 Binds directly to m7G cap

eIF-4G 220 Augments binding of eIF-4E to m7G cap; required for the
initial
round but not for sustained translation
eIF-4A 46 RNA-dependent ATPase; essential for binding of
ribosomes to
natural mRNA
eIF-4B 69 RNA binding protein; stimulates (but not essential for)
ribosome
binding to natural mRNA

eIF-5

45 Mediates hydrolysis of GTP associated with eIF-2 on 40S

subunit

(eIF-6 25kDa Ribosome anti-association factor)