Initiation of Translation

1
Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

2
Charging tRNAs With Amino Acids
Aminoacyl-tRNA synthetase enzymes carry out
two-step reaction
First, the amino acid is activated by reaction
with ATP to give an aminoacyl-AMP
Then the amino acid is transferred to the 3
end of the correct tRNA, with displacement of AMP
Fig. 17.2
3
The Ribosome Recognizes the tRNA, Not the Amino
Acid
tRNACys charged with Cys, then treated with
nickel-aluminum alloy (1962)
This gives desulfurization of Cys, converting
it to Ala
The newly formed alanyl-tRNACys allowed
incorporation of Ala into protein using Cys codon
Because there is no discrimination for the
amino acid by the ribosome, the aminoacyl-tRNA
synthetases must be highly specific
Fig. 19.28
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Extensive Interactions Between tRNAGln and Its
Aminoacyl tRNA Synthetase
Steitz and colleagues, 1989
Recognition occurs between the enzymes and
various parts of the tRNA

• acceptor stem (upstream from common region)
• Anticodon loop
• D stem

Class I enzymes, including glutaminyl-tRNA
synthetase shown, first aminoacylate the 2OH,
then rearrange the linkage to the 3 position
Class II enzymes immediately aminoacylate at
the 3 position
Aminoacyl tRNA synthetases also must recognize
the correct amino acid and exclude incorrect
amino acids.
Fig. 19.29
5
Exchange of Ribosomal Subunits
Mixture of labeled and unlabeled ribosomal
subunits established that they separate in vivo
Fig. 17.3
6
Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

7
A Fundamental Difference in ORF Structure
Between Prokaryotes and Eukaryotes
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IF3 Is Necessary For Irreversible Dissociation of
Ribosomal Subunits
Ochoa and colleagues extracted IF 1,2, and 3
from ribosomes with salt washes
They then added back the initiation factors to
ribosomes and used sucrose gradient
ultracentrifugation to monitor disassembly
Only IF3 by itself was able to promote
disassembly
IF1 is also involved in promoting disassembly,
and IF3 then binds to the 30S subunit to prevent
re-association
Fig. 17.7
Fig. 17.5
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Translation Starts Just Downstream From
Shine-Dalgarno Sequence
16S rRNA pairs with Shine-Dalgarno sequence
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The Initiator tRNA Is Charged With
N-formyl-methionine
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Steps of Translation Initiation In Prokaryotes
Fakunding and Hershey, 1973
Mixed 32PIF2, 3HfMet-tRNAfMet and AUG with
30S
Non-hydrolyzable analog GDPCP supported IF2
binding
Fig. 17.11
Fig. 17.15
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Steps of Translation Initiation In Prokaryotes
Without AUG and fMet-tRNAFMet, IF1 and IF3 are
required for stable binding of IF2
Fig. 17.15
Fig. 17.12
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Steps of Translation Initiation In Prokaryotes
Neither IF2 nor 70S ribosomes alone give
efficient GTP hydrolysis
Only in combination is strong hydrolysis
observed
GTP hydrolysis leads to removal of IF2
Fig. 17.15
Fig. 17.13
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Steps of Translation Initiation In Prokaryotes
GTP hydrolysis is required for release of IF2
Much more fMet-tRNAfMet bound and formed 70S
initiation complex in the presence of GTP
Fig. 17.15
Fig. 17.14
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Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

16
Most Eukaryotic Translation Is Cap Dependent And
Occurs By Scanning Mechanism
Eukaryotic initiation is with Met, not fMet.
But it still uses a different tRNA
Translation usually begins from the 5-most AUG
sequence
Scanning model developed by Marilyn Kozak
(1978) based on three things 1. Translation
was not known to begin from internal AUG 2.
Initiation did not occur at a fixed distance from
the 5 end 3. Translation is greatly
facilitated by the presence of the cap
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Effect of Multiple Copies of a Translation
Initiation Site
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Effects of Secondary Structure In 5 UTR On
Initiation of Translation
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Steps of Eukaryotic Translation Initiation
Algire and Lorsch, Curr. Opin. Chem. Biol. (2006)
10, 480-6.
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Stimulation of Translation By Cap-binding Protein
Sonenberg and colleagues used chemical
crosslinking to discover a protein complex bound
to the 5 cap of mRNA (1978)
Soon after cap-binding protein was discovered
(eIF4F complex), it was shown to stimulate
translation of capped mRNAs
Translation in HeLa extracts monitored by
incorporation of 35SMet
Fig. 17.24
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Components of Cap-binding Complex
eIF4F complex now known to contain three
proteins - eIF4E, which binds cap directly
- eIF4A, a DEAD-box protein - eIF4G, a large
protein (220 kDa) that binds several other
proteins
eIF4A has RNA unwinding activity and is
stimulated by eIF4B
Fig. 17.26
Fig. 17.25
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Role of eIF4G In Recruiting 40S Ribosomal Subunit
Adapter between eIF4E and eIF3, bound to 40S
Picrornovirus cleaves off N-terminal of eIF4G,
preventing interaction with eIF4E. eIF4G is still
functional in initiation, probably by bridging
IRES-binding protein and eIF3.
Binds Pab1p, resulting in circularization of
mRNA - protects RNA from degradation -
provides selection for full-length RNA - may
allow ribosomes to perform multiple cycles
of translation without dissociation
Fig. 17.27
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Internal Ribosomal Entry Sites (IRES) Found in
Some Viral RNAs
picornavirus encephalomyocarditis virus (EMCV)
43S particle recruited through direct
interaction of IRES and eIF4G
hepatitis C virus (HCV) direct, specific
recognition of 40S and eIF3 by IRES RNA
cricket paralysis virus (CrPV) associates
directly with 40S portion of IRES mimics tRNA in
P site
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Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

25
Small RNAs Regulate Translation in Prokaryotes
More than 40 sRNAs identified in E. coli
Only a handful have been characterized, but
these bind by base-pairing to mRNA targets and
regulate translation
DsrA RNA forms base pairs with rpoS mRNA, which
exposes Shine-Dalgarno sequence
rpoS encodes a stationary phase sigma factor
Some other sRNAs are known to repress
translation intead of activating
Fig. 17.35
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Riboswitches A Common Prokaryotic Mechanism of
Translation Regulation
Discovered by Breaker and colleagues (2002)
Shown is thiamine pyrophosphate (TPP)
riboswitch. Binding of TPP represses translation
of mRNAs that encode enzymes involved in TPP
synthesis
Biochemical studies performed to show clearly
that TPP binds to RNA element, inducing
conformational change that blocks Shine-Dalgarno
sequence
Riboswitches are ubiquitous in prokaryotes.
In addition to substances like vitamins, ligands
are - amino acids - nucleobases (guanine
and adenine) - sugars - metal ions
Fig. 17.36
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Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

28
Phosphorylation of Initiation Factor eIF2?
Used extensively in reticulocytes, which
basically make only hemoglobin
If cell is starved for iron, heme-controlled
repressor phosphorylates one of the subunits of
eIF2, known as eIF2?
Phosphorylated eIF2 binds eIF2B tightly, which
sequesters eIF2B such that it is unable to
promote GDP/GTP exchange
This leads to global repression of translation
Fig. 17.37
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Regulation by an RNA Element
Munro, Klausner, and colleagues demonstrated
repression of translation by RNA sequence (1987)
Repressor protein binds IRE element in 5 UTR
until removed by Fe2
When Fe2 level is high, increased translation
of protein ferritin allows for increased iron
storage.
Assayed using chloramphenicol acetyltransferase
(CAT) reporter gene and monitoring acetylation of
chloramphenicol using TLC system
Lacked IRE
Fig. 17.45
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More Recent Structural Work Has Further
Elucidated Mechanism
NMR showed structural changes in element of RNA
in 5 UTR upon binding of repressor IRP1 (also
shown to be aconitase).
Stem structure is only 20 nt from 5 end,
suggesting that repression is mediated by
preventing the 40S subunit from binding.
Repressor protein, aconitase, also binds Fe-S
cluster. Binding of this cluster leads to a
conformational change which prevents binding to
IRE
Leipuviene and Theil, Cell. Mol. Life Sci. (2007)
64, 2945-2955
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miRNA A Major Pathway For Translational
Regulation in Eukaryotes
We will hear much more about micro RNAs
(miRNAs) in two weeks
Fig. 16.45
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Key Points
1. Prokaryotic and eukaryotic translation
initiation have some common features. A charged
initiator tRNA forms a complex with the small
subunit that recognizes a start codon, followed
by joining of the large subunit, GTP hydrolysis,
and ultimately initiation.
2. Eukaryotic initiation has unique features
most is cap-dependent and occurs by scanning, the
mRNA circularizes, and there are more factors.
Some mRNAs (especially viral) use IRES elements
3. Prokaryotes and eukaryotes have varied
mechanisms for control of translation. These make
extensive use of RNA-RNA interactions and RNA
structures.
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