The document summarizes eukaryotic translation initiation. It describes how the 43S preinitiation complex is formed and recruits to the 5' end of mRNA with the help of initiation factors. The complex then scans the 5' UTR until it recognizes the start codon, after which the 60S subunit joins to form the 80S ribosome. Initiation factors are regulated by phosphorylation and proteolysis. Translation can also be controlled by RNA-binding proteins and the length of the poly-A tail.

1. Presented by: NIDA REHMAN
university of karachi, (pakistan)

2. Main focus:
Eukaryotic initiation complex.
Factors involve in the eukaryotic translation.
Control of initiation factors

3. Overview:
 Translation, the process of mRNA-encoded protein
synthesis, requires a complex apparatus, composed of the
ribosome, tRNAs and additional protein factors, including the
initiation factors.
 The ribosomes are ribonucleoprotein particles to which
multiple ribosomal proteins are bound. The sequence and
structure of ribosomal components are conserved in all
kingdoms, underlying the common origin of the translation
apparatus.
 The ribosome provides the platform for proper assembly of
mRNA, tRNAs and protein factors. It consists of small and
large subunits.

4.  Translation can be subdivided into several steps :
1. Initiation
2. Elongation
3. Termination
4. Recycling
 Of these, initiation is the most complex and the most divergent among
the different kingdoms of life. A great amount of new structural,
biochemical and genetic information on translation initiation has been
accumulated in recent years, which led to the realization that initiation
also shows a great degree of conservation throughout evolution.
 Translation initiation in eukaryotes is a highly regulated and
complex stage of gene expression. It requires the action of
at least 12 initiation factors, many of which are known to be
the targets of regulatory pathways. Here we review our
current understanding of the molecular mechanics of
eukaryotic translation initiation.

5. Translation Mechanism
 In the translation mechanism, diverse proteins known as
translational factors are involved converting the information
contained in the mRNA into a protein. This event is
commonly divided into three phases: initiation, elongation,
and termination
 The initiation phase has been described as the most
regulated in eukaryotic cells, it can be carried out in two
ways:
 (a) cap-dependent or conventional and
 (b) cap-independent or internal ribosome entry site (IRES).

6. Cap-Dependent Translation Initiation
 Most mRNAs of the cell are characterized by the m7GpppX structure
(where X is any nucleotide) at the 5′ end, called cap. The 3′ end of mRNA
contains a polyadenylated tract (poly-A), which is attached to the poly-A-
binding protein (PABP). Both cap and poly-A have been observed to play
key roles in translation efficiency
 In the cap-dependent mechanism, to translate an mRNA, it is important
that the mRNAs joining to a protein complex called eIF4F.
 43S is another complex involved in the initiation phase. 43S is formed by
the small ribosomal subunit 40S, the factor eIF3, and ternary complex. It
has been proposed that the eIF4F complex join the 43S complex via the
interaction between eIF3 and eIF4G.
 The ribosomal subunit 40S carries the eIF2-GTP-Met-tRNAi complex to the start
codon, where both ribosomal subunits 40S and 60S blend to form the complete
ribosome 80S, eIF2 is released along with GDP. The GDP bound to eIF2 is
exchanged for GTP by the eIF2B factor to start a new initiation round.

7. Initiation complex. The interaction between the eIF4F complex, 43S, and
mRNA is shown. EIF4F is formed by eIF4A, eIF4G, and eIF4E. The complex
43S is formed by eIF3, the small ribosomal subunit, and eIF2, which in turn is
formed by methionine-tRNA-initiator (Met-tRNAi) and GTP. The mRNA is
recruited to the eIF4F complex across the interaction of the 3′ end and poly-A-
binding protein (PABP) and the 5′ cap and eIF4E. UTR: untranslated region.

8. Cap-Independent Translation Initiation
 The cap-independent translation mechanism occurs when cap-
dependent translation is limited.
 This alternate initiation proposes that a complex secondary structure in
the 5′ untranslated region (5′ UTR) called IRES is important for
translation of mRNA.
 The IRES mechanism was initially described in the picornavirus family.
 However, it is now known that the IRES is not unique to viral mRNAs, as
it has been found that IRES-containing cellular mRNAs code principally
for proteins involved in cell recovery from stress conditions and during
the cell cycle.
 Interestingly, in this initiation form, an mRNA with IRES can be translated
with or without the requirement of any canonical initiation factor or can
use cellular proteins known as IRES trans-acting factors (ITAFs) that
function as cofactors to facilitate or encourage translation.

12. Eukaryotic translation initiation:
1.Formation of the 43S pre-initiation complex,
when the Met-tRNA is delivered by eIF2 to the
P site of the 40S ribosomal subunit;
2.Recruitment of the 43S complex to the 5' end
of the mRNA by eIF3 and the eIF4 factors;
3.Scanning of the 5' untranslated region (UTR)
and recognition of the AUG codon, and
4.Assembly of the 80S ribosome

13.  Recycling of post terminational complex to yield separate
40S and 60S subunit, result in the formation of 80S
ribosomal initiation complex, in which Met-tRNA base
paired with the initiation codon in the ribosomal P site and
which is competent to start the translation elongation state.
 eIF2-GTP-Met-tRNA ternary complex formation.
 Formation of preinitiation complex (40S subunit,eIF1,
eIF1A, eIF3,eIF2-GTP-Met-tRNA & eIF5
Pathway of translation
initiation in eukaryotes

14.  mRNA activation, during which mRNA cap proximal
region is unwound in an ATP dependent manner by eIF4F
with eIF4B
 Attachment of 43Scomplex with mRNA region
 Scaning of 5’ UTR in 5’ to 3’ direction by 43S complex
 Recognition of initiation codon and the formation of 48S
complexes, which switches the scaning complex into
closed conformation and leads to the displacement of
eIF1 to allow eIF5- mediated hydrolysis of eIF2 bound
GTP and P release
 Joining of 60S subunit to 48S complex, formation of 80S
subunit.

15. Pathway of translation initiation in eukaryotes
Klann and Dever, Nature Reviews (2004
o A binary complex of eukaryotic
translation initiation factor 2 (eIF2) and
GTP binds to methionyl-transfer RNA
(Met–tRNAMet)
o This ternary complex associates with
the 40S ribosomal subunito Association of additional factors, such
as eif3 and eif1a (1A), with the 40S
subunit promotes ternary complex
binding and generates a 43S pre-
initiation complex. The cap-binding
complex, which consists of eif4e(4E),
eIF4G and eIF4A (4A), binds to the 7-
methyl-GTP (m7GTP) cap structure at
the 5’end of a messenger RNA (mRNA).o eIF4G also binds to the poly(A)-binding
protein (PABP), thereby bridging the 5’
and 3’ ends of the mRNA.
o Following scanning of the ribosome to
the AUG start codon, GTP is hydrolyzed
by eIF2, which triggers the dissociation
of factors from the 48S complex and
allows the eIF5B- and GTP-dependent
binding of the large, 60S ribosomal

18. Mechanism of 5’ end dependent initiation:
 Formation of 43S preinitiation complex.
 Attachment of 43S complex to mRNA.
 Ribosomal scanning of mRNA 5’ UTR
 Initiation codon recognition
 Commitment of ribosome to a start codon.
 Ribosomal subunit joining.

19. Formation of 43S preinitiation complex
 Translation is cyclic process, for recycling of ribosomal subunits low Mg2+
concentrationis required.
 Recycling can be mediated by eIFs (eIF3, eIF3j, eIF1, eIF1A)
 eIF1 & eIF1A dissociates post termination ribosomal complex into 60S
subunit and mRNA and tRNA bound 40S subunit.
 eIF1 release tRNA after which eIF3j mediated mRNA dissociation.
 eIF1 and eIF1A remain associated with 40S subunits preventing their
re-association, eIF6 bind to 60S subunit to prevent re-association.
 Recycling also requires ABCE1, also help in the splitting of ribosome.

20.  Prokaryotic and eukaryotic ribosome share a common
structural feature, the structural homology between
eukaryotic and prokaryotic ribosomes allow the use of
high- resolution of crystal structure of prokaryotes
ribosomes to model 40S-eIF interactions.
 40S subunit consist of head, a platform and a body, with
mRNA binding channel.
 Binding of eIF1 and eIF1A to 40S subunit induces
conformational changes, which involve the opening of
mRNA opening channel.

21. Model of 40S subunit with eIF3
on it’s exterior surface and
eIF4G bound to eIF3 near the E
site. Also showing the position
of mRNA and eIF1 on the
subunit interface.
• Position of eIF1 and eIF1A on a
40S subunits.
• eIF1 is in magenta
• eIF1A (structural domain is in light
blue, carboxy-terminal tail in dark
blue, and amino terminal tail in
green)
• mRNA is in red color.
• tRNA yellow color.

22. Apo 40S subunit and 40S-eIF1-eIF1A complexes, on the right hand side
A, P and E site of ribosome is labeled in the mRNA binding channel and
position of rRNA helicases which are involved in forming mRNA entry
channel and eIF1 and eIF1A induced head shoulder connection

23. Attachment of 43S complex to mRNA
 5’UTR posses sufficient secondary structure for loading of
43S complex.
 It requires eIF4F, eIF4B or eIF4H, which unwind 5’ proximal
region prepare it for ribosomal attachment.
 eIF4B and eIF4H enhance eIF4A helicase activity. (eIF4H
contain RRM homologous to eIF4A, also prevent
reannealing of mRNA and promoting eIF4A unidirectional
movement)
 5’ cap binds to eIF4E ,concave surface by stack between

24.  eIF4A has 2 domain, both domains has a contiguous RNA-
binding surface and ATP binding site. The activity of eIF4A
depend upon eIF4G and eIF4B.
 eIF4A eventually dissociates from mRNA but being anchored
to it’s 5’ end by eIF4E cap interaction, this complex resume
another cycle of unwinding, thereby keeping 5’ proximal
region constantly available prepared for ribosome
attachment, this ribosomal attachment facilitated by eIF3-
eIf4G interaction.
 The requirement of 43S complex is achieved by cap-eIF4E-
eIF4G-eIF3-40S chain of interactions.
 The open latch conformation of 40S subunits, induced by
eIF1 and eIF1A, is likely to be strongly conductive for
attachment.

25. Ribosome scanning of mRNA 5’ UTRs
 After attachment to the 43S complexes scan mRNAs downstream
of a cap to initiation codon.
 Scanning consist of 2 linked process
 Unwinding of secondary structure at 5’ UTR
 Ribosomal movement along it.
 43S complex scan unstructured 5’UTRs without factors associated
RNA unwinding and are intrinsically capable of movement along
mRNA
 Scanning at 5’ UTR containing weak secondary structure requires
ATP and eIF4A, eIF4G, and eIF4B.

26. Initiation codon recognition:
 The 1st AUG triplet in an optimum context –
GCC(A/G)CCAUGG, with a purine at -3 and a G at+4
position.
 The A of the AUG codon designated as +1
 eIF1 play a key part in the fidelity of initiation.
 eIF1 enables the 43S complexes to discriminate against
non AUG triplets and AUG triplets that have poor
context.
 Codon anticodon base pairing is established by
tightening of eIF1A- 40S interactions .
 The purines at -3 and+4 positions probably affect
initiation codon selection.

27. Ribosomal subunit joining:
 Joining of 60S subunits and dissociation of
eIF1,eIF1A, eIF3 and a residual eIF2-GDP are
mediated by eIF5B
 Hydrolysis of eIF5B –bound GTP is not
required for subunit joining, but it’s essential
for eIF5B own release from assembled 80S
ribosome

28. Control of initiation factors
 Mechanisms of regulating initiation fall into two
broad categories:
I. mechanisms that impact on the eIFs (or
ribosomes),
II. those that impact on the mRNA itself,
a. RNA-binding proteins
b. microRNAs (miRNAs)
 The initiation factor 2 & 4F is also control by rversible
protein phosphorylation, eIF4F is also control by
irreversible protein proteolysis of eIF4G

29.  There are 4 protein kinases that phosphorylate eIF2a on
Ser51.
i. Haem regulated kinase
ii. Protein kinases RNA-activated (PKR)
iii. Protein endoplasmic reticulum kinases (PERK)
iv. General control nonderepressable 2 (Gcn2)
 Phosphorylated eIF2 form an initiation competent eIF2-TC,
but the phosphorylated eIF2-GDP tightly binds eIF2b nullify
its activity.
 eIF2-TC level fall and most mRNA translation is reduced, but
those who have two uORFs in appropriate type and position
can actually be stimulated.
 Example: ATF4 & ATF5 expression is increased 5 fold by
activation of PERK
 Phosphorylation also affects intracellular concentration of
eIF.
 Phosphorylation of eIFs increases under condition in which
translation is activated

31. Regulation by RNA binding protein:
 Inhibitory effect on translation except
PABP.
 For translation these protein must be
degraded.

32. Regulation by specific 5’ UTR protein interaction:
 Rare but just one example; ferritin mRNA (iron).
 For strong inhibition of translation requires the protein
RNA interaction at cap proximal location, prevent
loading of 43S complex onto mRNA.
 Inhibition is much weaker if protein binding RNA motif
moved to more cap distal position.
 In this 43S complex loaded over the mRNA, it’s
subsequent scanning will displace the bound protein.

33. Stimulation by 3’ poly A tail:
 PABP have stimulatory effect.
 PABPs second RRM domain interact with eIF4G, result
in the circularization of mRNA in the closed loop
configuration.
 By the help of PABP eIF4F remain anchor to mRNA at 3’
poly A tail, this anchoring will not be occur in the
absence of PABP or poly A tail.

34. Regulation by specific 3’ UTR protein interaction:
 Control by 3’ UTR is entirely dependent on changes in
poly A tail length, because the regulatory mRNAs are
repressed when they have a short tail or activated when
they have a long length.
 in some cases translation can be activated with out the
lengthening of short poly A tail, in some cases protein
may changes the polyadenylation status.

35. Protein X binds in a sequence-specific manner to a specific 3′ UTR motif of mRNA
and interacts with an intermediate bridging protein (protein Y), which in turn interacts
with a cap-binding protein (protein Z), leading to the formation of an inhibitory closed
loop that precludes access of eukaryotic initiation factor 4F (eIF4F) to the 5′ end (see
the figure). As protein X is the only sequence-specific RNA-binding protein amongst
the three, the identity of protein X in the complex differs more widely between
different mRNAs or groups of mRNAs than the identities of protein Y and protein Z
(see the table). The functions of protein X and protein Y can be embodied in a single
protein (for example, Bicoid) or in a group of proteins (Nanos, Pumilio and Brat)86. It
should be noted that although maskin has been claimed to be protein Y in Xenopus
laevis oocytes121, its interactions with cytoplasmic polyadenylation element-binding
protein (CPEB) have not been seen in some laboratories87, the motif by which it is
supposed to interact with eIF4E is not conserved in maskins from other species, and
it is only expressed in the late stages of oogenesis87.

36. Translation regulation by miRNAs:
 Sequence specific
 Repress translation at 3’ UTR
 Can act in conjunction with RNA binding protein
 Almost 21 nucleotide
 Degree of repression increases with the increasing number of
miRNA
 Repression efficiency might also be influenced by the distance and
sequence between miRNA target sites and also their position in the
3’ UTR
 In some cases miRNA act as a adaptor for sequence specific RNA
binding protein.

37.  The mechanism of repression have 2 main component
 Normal mRNA translation
 Deadenylation dependent pathway
 Repressed mRNA displace from large polysome to small polysome
or sub polysomal partical, this indicate the inhibition of initiation.