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 don’t

ribosomes initiate translation at other
AUG’s?
• 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
5’ GCC G CCAUGG 3’

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
AUG’s (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