MB3003 Eukaryotic Translation

Prof Anne Glover (l.a.glover@abdn.ac.uk)

Lecture 1: Translation initiation

Relevance of translation
initiation
Another level of control of gene
expression
Is there a common theme?

Phases of translation
1. Initiation
2. Elongation
3. Termination

Identifying the right place

on the mRNA

In prokaryotes, mRNA is polycistronic

Translation starts (primarily) at AUG

AUG codes for methionine - why dont
ribosomes initiate translation at other
AUGs?
Prokaryotes - Shine-Dalgarno sequence
directs ribosomal subunit to initiation
codon
Shine-Dalgarno sequence is
complementary to sequence on 16S
ribosomal subunit

Shine-Dalgarno sequence
mRNA

5-UAAGGAGG-(5-10 nucl)-AUG
OH-AUUCCUCC-(~1400 nucl)-5

rRNA

Interaction between sequences in mRNA (near

translation start site) and 16S rRNA (near 3
end)
Bacterial ribosomes can initiate translation at
these sites in middle of long RNA transcript,
i.e. polycistronic RNA

Prokaryotic translation initiation

needs additional factors
As well as the Shine-Dalgarno
sequence, additional proteins factors
(initiation factors) are required
Called IF1, IF2 and IF3
Recognise AUG, small ribosomal
subunit, other Ifs and initiating tRNA

Fundamental difference in initiation between

prokaryotic and eukaryotic translation
In prokaryotes, the small ribosomal subunit
recognises the Shine-Dalgarno sequence of
the mRNA these can be internal
In eukaryotes, the small ribosomal subunit
recognises the 5 cap structure on the
mRNA and translation initiates from the
closest AUG

Translation initiation in eukaryotes

1. Absolute requirement for cap on 5 end
(majority of RNAs)
2. For 95% of eukaryotic mRNAs translation
begins at 5-proximal AUG
3. Small ribosome subunit can scan in one
dimension on RNA
4. mRNA secondary structures located in the
5-untranslated region inhibit translation
initiation

Kozak consensus sequence

A
GCC G CCAUGG

Marilyn Kozak identified this consensus

sequence around the AUG start codon.
It enhances initiation frequency

Eukaryotic initiation of translation

Precise sequence of events

Lodish Fig 4-37

Characteristics of mammalian translation factors

Name

Function

eIF1A

Promotes Met-tRNA binding, ribosomal dissociation

eIF2

Binds Met-tRNA and GTP

eIF2

Site of phosphorylation on Ser-51

eIF2B

Guanine nucleotide exchange factor for eIF2

eIF3

Dissociates ribosomes, promotes Met-tRNA and mRNA binding

eIF4A

ATPase, helicase, binds RNA

eIF4B

Binds RNA, promotes helicase activity

eIF4E

Cap-binding subunit, part of eIF4F complex

eIF4G

Binds eIF4A, eIF4E and eIF3 acts as bridging factor

eIF5

Promotes GTPase with eIF2 and ejection of eIFs

eIF6

Binds to 60S ribosomes, promotes dissociation

Stages in translation initiation

1. eIF3 binds 40S ribosome - eIF6 binds 60S
ribosome to keep them apart
2. eIF2 binds GTP + initiating methionine tRNA
to form eIF2.GTP.mettRNA. This binds to 40S
3. RNA is loaded with factors at cap site;
eIF4E (cap binding protein), eIF4B and eIF4A
(unwind RNA)
4. 40S ribosome (+ eIF3 + ternary complex)
binds to cap (interaction of eIF3 with eIF4G)

5. 40S ribosome scans along 5-UTR (requires

ATP)
6. When AUG is reached, eIF2.GTP is
hydrolysed (possibly triggered by eIF5) which
releases all factors from 40S subunit
7. 60S subunit can then bind to 40S subunit
8. eIF2.GDP must be recycled to eIF2.GTP by
action of eIF2B

Cap-independent translation initiation

Picornavirus mRNAs have no cap at 5-end
Instead have long 5-UTR with many upstream
AUGs (normally inhibits translation)
Translation of picornavirus RNA v. efficient even if inhibit cap-dependent translation
RNAs contain internal sequence that recruits
ribosomes
Termed IRES - internal ribosome entry site

Features of IRES allowing ribosome binding

Generally long (450 nt for picornavirus)
Lack identifiable consensus sequence - Py tract
in picornavirus (not in cellular RNAs)
Some complementarity to 18S rRNA
Likely tertiary structure is important

Mechanism of internal initiation

Requires same factors as cap-dependent
initiation EXCEPT FOR eIF4E
Other protein factors may help assemble
correct RNA tertiary structure

Eukaryotic translation initiation can occur at

IRES

Lodish Fig 4-38

Viral inhibition of cellular translation

Some viruses (adenovirus and influenza virus)
cause dephosphorylation of eIF4E
Others e.g. picornavirus cleave eIF4G into 2
fragments
C-terminal fragment still interacts with eIF3
and eIF4A
N-terminal fragment still interacts with eIF4E
Mechanism of shutting down host cell translation
while maintaining their own