The Initiation of Translation in Pro and Eukaryotic Cells

1
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.
2
Eu- and Prokaryotic Ribosomes
Eukaryotic cytoplasm
Prokaryotes, Eukaryotic organelles
(mitochondria, chloroplasts)
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E, P and A Sites of Ribosomes
E Exit site for free tRNA P peptidyl-tRNA
A aminoacyl-tRNA
4
Initiation of Translation in Prokaryotes
IF-2 initiation factor 2 In complex with GTP,
it brings fMet-tRNAfMet 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.
50S
IF-3
RRF
50S


mRNA
30S
30S
IF-2?GTP?fMet-tRNAfmet
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

mRNA
(fMet-tRNAfMet)
GTP
2
30S Initiation Complex
5
30S Initiation Complex

GTP
2
GTP
2
GDP Pi
2
Asite
Psite
70S Initiation Complex
Elongation Phase of Translation
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Initiation of Translation in Prokaryotes
Simple process involves only initiation factors
(IFs) IF-1, IF-2 and IF-3
plus.. fMet-tRNAfMet 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
8
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.
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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 .
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Prokaryotic mRNAs may be polycistronic
sites of ribosome re-cycling
cistron 3
cistron 1
cistron 2

• 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)

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Initiation of Translation in Prokaryotes
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Initiation of translation in Eukaryotes
Its (much) more complex ..
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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
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Scanning Model of Eukaryotic Initiation of
Translation
(initiating) AUG
5 cap
Stop codon
open reading frame
A(n)
me7Gppp
43S
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(initiating) AUG
5 cap
Stop codon
scans
open reading frame
A(n)
me7Gppp
16
(initiating) AUG
5 cap
Stop codon
A(n)
me7Gppp
17
(initiating) AUG
5 cap
Stop codon
A(n)
me7Gppp
60S
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(initiating) AUG
5 cap
Stop codon
A(n)
me7Gppp
Elongation Phase
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Messenger RNA structure
Open reading frame
Cap
Poly(A) tail
AUG
stop
AAAAAAAAAAAAAAAA
m7Gppp
5NCR
3NCR
Exon / exon splice boundaries
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Messenger RNA structure 5 NCR
RNA stem-loop structures
AUG
m7Gppp
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Messenger RNA structure 3 NCR
A / U rich Elements (AREs) - binding
sites for stabilising
/ destabilising proteins
stop
orf
AAAAAAAAAAAAA
mRNA localisation elements (usually located in
the 3NCR) - 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
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Initiation of Translation
eIF6
60S
60S
60S
40S
6

Ribosome anti-association factor
40S
eIF3
40S
Binds to 40S subunit (stabilises Met-tRNAi) and
prevents association with 60S subunit
3
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Ternary complex formation
GDP-bound eIF2 cannot bind Met-tRNAiMet
guanine nucleotide exchange factor (GEF)
initiator methionyl tRNA
ternary complex
eIF3
GTPase activating protein (GAP)
eIF3
multifactor complex (MFC)
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43S Complex Formation
multifactor complex (MFC)
eIF1A and eIF3 promote binding of the multifactor
complex to the 40S subunit
40S
43S Complex
eIF3
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Assembly of the Cap Binding Complex Eukaryotic
Initiation Factor 4G (eIF4G)
Binds poly(A) Tails
40S
Binds 7meG caps
eIF3
eIF4E
PABP
eIF4A
eIF4A
eIF4G
Binds eIF3
eIF4E cap binding eIF4A bi-directional RNA
helicase MnkI MAP kinase-interacting protein
kinase-1 (phosphorylates eIF3) PABP poly(A)
binding protein
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Recruitment of the 43S complex to the 5 end of
the mRNA
AAAAAAAAA
40S
PABP
PABP
eIF3
eIF4G
eIF4E
eIF4B
eIF4A
m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

• eIF3 interacts with eIF4G to recruit the 43S
  complex
• eIF4B RNA binding protein stimulates (but not
  essential for) ribosome
• binding to natural mRNA

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Scanning of the 5 UTR and AUG recognition
ATP
ADP Pi
eIF4G
cap-binding complex recycled
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Conformational change, GTP hydrolysis, release
of initiation factors, and assembly of the eIF5B
GTPase
40S
eIF5
eIF3
eIF1
eIF1A
m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC
GTP hydrolysis by eIF2 requires eIF5 and
possibly a conformational change triggered by
the Met-tRNAiMet interaction with the 40S subunit
40S
Met
GTP
eIF5B
eIF1A
m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC
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Assembly of the 80S ribosome
40S
GTP hydrolysis by eIF5B serves as a
final checkpoint for correct 80S assembly
eIF5B
m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC
60S
6
40S
eIF5B
6
m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC
60S
the 80S ribosome is now poised to elongate
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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
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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 (a), 38 (b), 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)
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The Elongation Phase of Translation
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Elongation Cycle of Eukaryotic Protein Synthesis
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
EF1A ? GTP
An
5'
An
5'
A
P
A
P
P

EF2 ? GDP
EF1A ? GTP
aminoacyl-tRNA binding
Translocation
aa
EF1B???
aa
aa
EF2 ? GTP
aa
aa
peptidyl transfer
aa
aa
aa
aa
aa
EF1A ? GDP
P
5'
An
An
5'
A
P
A
P
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Elongation factor 2 - a molecular motor
tRNA
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The Termination Phase of Translation
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- GGQ -
CCA acceptor stem
Human eRF1
tRNA
anticodon-like site -TASNIKS-
anti-codon loop

• terminates translation
• recognises all three stop codons
• activates a water molecule to hydrolyse
  tRNA-peptide ester linkage

eRF1
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eRF3 a molecular mimic of elongation factor 1A
eRF3
EF-tu (eEF1A)
GTPase enhances termination efficiency stimulat
es eRF1 activity in a GTP-dependent manner
tRNA
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eRF3 binds and accelerates the dissociation of
eRF1 - nascent protein released
eRF1 binds into A site (interacts with stop
codon)
eRF1
G
H20
G
G
OH
OH
OH
OH
OH
E P A
E P A
E P A
eRF1 activates a water molecule to hydrolyse
the peptidyl-tRNA ester linkage
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