The mechanism of eukaryotic translation

initiation and principle of it’s regulation.

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

Main focus:

Eukaryotic initiation complex.Factors involve in the eukaryotic

translation.Control of initiation factors

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.

Translation can be subdivided into several steps : 1. Initiation2. 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.

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).

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.

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.

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.

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

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 translationinitiation in eukaryotes

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.

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 subunit

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.

Formation of 43S preinitiation complex

Translation is cyclic process, for recycling of ribosomal subunits low Mg2+ concentration is 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.

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.

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.

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

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 two trptophan residues of eIF4E.

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.

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.

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.

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

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

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

Regulation by RNA binding protein:

Inhibitory effect on translation except PABP.

For translation these protein must be degraded.

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.

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.

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.

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.

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.

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.