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Eukaryotic Translation INITIATION - 80S ribosome assembly steps | Initiation factors in mRNA loading

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Summary

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This content details the intricate process of eukaryotic mRNA translation initiation, focusing on the assembly of the 40S ribosomal subunit with initiation factors and mRNA, followed by the recruitment and joining of the 60S subunit to form the functional 80S ribosome.

Mind Map

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Before we dive into the process, there

are two things that you should keep in

mind. The first is the structure of the

ukarotic ribosome which consists of two

subunits. The 60S and the 40S subunit.

The 60S is made up of proteins and three

major ribosomal RNAs. These are 28S,

5.8S, and the 5S ribosomal RNA. On the

other hand, the 40S subunit has the 18S

ribosomal RNA as its major component.

And this entire protein and RNA complex

in ukarots is known as the ATS ribosome.

And the processing of the mRNA and the

synthesis of the polyeptides occurs at

these so-called EP and A sites in these

ATS ribosomes. And we discussed these

sites earlier in the translation series.

So I will draw out the ribosome like

these dome-shaped structures. But always

keep in mind that over 60% of the

ribosome is made up of ribosomal RNAs.

The second important point is the

structure of the ukareotic mRNA. The

ukareotic mRNA begins with a fivep prime

utr which often times contains secondary

structures like hairpins and stem loops.

The fivep prime UTR typically also has a

ribosome binding site which in ukarots

is known as the coac sequence. This

fivep prime UTR is followed by the

coding sequence which starts at the

start codon and this coding frame

continues until a stop codon is

encountered and towards the end of the

coding sequence you have the threep

prime utr which is followed by a poly aa

tail. The fivep prime end of most all

ukarotic mrnas have a fivep prime

modification. You can learn all about

these modification in the transcription

videos. The links are down in the

description as well as on the cards

above. Now these modifications at the

threep prime and the fivep prime end

recruit some specific factors or

proteins that cause the mRNA to take a

loop structure such that the fivep prime

and the threep prime ends come close to

each other. We touched on this a little

bit when we were discussing the

transcription in ukarots and we'll see

later in this video how these

interactions are useful for the process

of translation initiation. The

initiation process can begin when the

two subunits, the 60S and the 40S are

not in the 80s ribosome form. This could

be either when ribosome is being

recycled from a termination step or when

a new subunit is being assembled. So the

point is that the 60S subunit usually

has to wait because the initiation of

translation begins with the 40S subunit.

And this is exactly what we saw in the

procariats where it started with the 30S

subunit. By the way, I will point out

some similarities and differences

between proaryot and ukarotic

translation initiation as we move along

in this video. So I hope you've watched

the previous videos. So the first factor

to associate with the 4DS small subunit

is the ukareotic initiation factor 1

which binds at the interface region

between the E and the P site and later

in the process one side of this

interface becomes the E site and the

other side becomes the P site. This Eiff

is similar to IF3 that we saw in the

proarotic translation. On the other end

of the 4DS subunit, the A site is

defined by the binding of Eiffa.

And this factor is very similar to the

initiation factor 1 in procaryots. The

middle ground between these two factors

is mostly the P site. Now all these

interactions that occur on the 40S

subunit are mediated by a larger

ukareotic initiation factor known as EIF-3.

EIF-3.

This protein is almost as big as the 40S

subunit itself. The C terminal end of

this EIF3 is pretty close to the A site

and the N terminal domain is located

pretty close to the E site. Now let's

talk about the functionality of these

factors. The EIF-1A primarily blocks the

tRNA binding to the A site and one

additional function that it serves which

we will see later is that it also helps

in the docking of the 60S subunit. The

EIF-1 on the other hand prevents the 60S

subunit from joining. And there's one

more function that EIF-1 serves which is

that it positions the mRNA at the 40S

subunit. And this large initiation

factor 3 as we said is a scaffold

protein which interacts with most all

initiation factors to regulate the

overall 4DS subunit interactions. The

one interaction regarding EIF-13 that I

would like you to note is that the

initiation factor 3 subunit J also

called EIF3J which is part of the C

terminal domain contacts the 4DS subunit

to define the mRNA entry channel. So

after these three initiation factors

assemble, the 4DS subunit is ready for

the next step. In this step, the

initiator tRNA which almost always

carries the methionine is bound by the

initiation factor 2. And this eif2 is

attached to a GTP. This charged

initiator tRNA is different from the

proarotic tRNA in that there is no form

modification done to the methionine. And

the function of EIF2 is similar to the

function of initiation factor 2 in

procaryots which also helps bind the

initiator tRNA and helps it to recruit

onto the 30th subunit. And for your

note, this anti-codon on this activated

tRNA must be UAC. If it is not obvious

why, it'll become clear in the next

step. So let's just quickly complete the

sketch of this 4DS complex where we have

the three initiation factors bound. Now

this initiator tRNA which contains the

methionine is brought to the pite with

the help of ukarotic initiation factor 2

and this eif2 interacts with both eif

and eif1a

and this helps in the positioning of the

initiator tRNA. Once ukareotic

initiation factor 2 has positioned,

another initiation factor called eif5

binds to eif-1 and eif2 to increase the

affinity of this complex. And on top of

this, the ukareotic initiation factor 5

also has a gtpas activating domain. So

this is the complex now where you have

EIF-1 at the E and P site transition the

initiator tRNA at the P site and the

EIF-1A at the A site. So this 40S

complex where all these initiation

factors are bound along with the

initiator tRNA. But there's no mRNA

bound yet is called the 43S

pre-initiation complex. And once this

43S pre-initiation complex is formed and

the interaction between IF2, EIF-1 and

EIF-1A is stabilized, the EIF-1 and

EIF-1A cause a structural change in the

40S subunit such that the mRNA entry

channel now opens up, which means that

this mRNA is ready to be bound onto the

43S complex.

But this mRNA has to undergo specific

steps before it can get recruited. And

these steps involve the interaction of

the fivep prime and threep prime end of

the mRNA. We quickly glossed over this

step earlier, but now let's get a bit

concrete and look at the details. The

ukareotic mRNA has a fivep prime cap

modification which is recognized by a

protein called ukareotic initiation

factor 4E. The mRNA on the other end has

a polya tail which is bound by

stabilizing proteins known as polya

binding protein C1. These polya binding

proteins recruit initiation factor 4g.

But eif4g not only has affinity for

polya binding proteins. It also has

affinity towards the eif4e which is

located at the fivep prime end. So in

principle no matter where it gets

recruited either at five prime end or

threep prime end the two ends later will

come together to form a loop regardless

but let's just say for the sake of

simplicity that it gets recruited at the

polyatail the e-4g then recruits eif4a

which happens to be a dead box hilicase

and now this mRNA after binding the

initiation factors at the fivep prime

end and the threep prime end can form a

loop such that the initiation ation

factor 4 E and 4G are brought together

due to the capping and the polyatail

modifications on the mRNA. The ukareotic

initiation factor 4 A which is the

helilicase by itself has very little

activation energy. So to help this

factor another protein is required which

is called initiation factor 4B and

simply put EIF4B enhances the EIF4A

helilicase activity. Now this complex of

initiation factors bound to the mRNA

become active and as a result the

helilicase EF4A moves forward by

consuming ATP as its energy source and

when it moves it unwinds the secondary

structure like hairpins and stem loops

in the five prime UTR and this unwinding

may be required to make the ribosome

binding site more accessible. So if we

sketch out the resulting structure, we

see that the hilicase has moved forward

and displaced the secondary structure.

So important thing to keep in mind here

is the role of hilicase in breaking the

five prime UTR hairpins. To make things

easier for remembering all this, note

that initiation factors that bind to the

mRNA are named 4E, 4G, 4 A, and 4B.

Essentially, this is saying that

initiation factors in class 4 are

involved in the mRNA binding. And after

binding, they help in the loading of the

mRNA onto the 43S pre-initiation

complex. Now that this mRNA is ready for

loading, let's see how this structure

would look after loading. I'm going to

draw out the 43S pre-initiation complex

first. And while I complete the sketch,

you can go ahead and subscribe to the

channel if you haven't already so you

don't miss out on the upcoming videos.

Don't be shy. Subscribing is totally

free. All right. Now for loading of the

mRNA onto the ribosome, the EIF4 complex

on the mRNA is recruited near the A site

on the small subunit. So if you draw out

this complex EIF4 on the 4DS subunit,

you notice that EIF4A is closely

associated to the EIF-1 near the E site.

And the mRNA is threaded through this

complex into the mRNA entry channel and

looped out such that the threep prime

end and the fivep prime end are

physically in contact as we have seen

already. Note that we said the initiator

has the anti-codon UAC which means that

its complimentary sequence is the AUG

which is the start codon on the mRNA.

But the helilicase activity of EIF4A was

simply done to unwind the hairpin so

that it can bind the ribosome which

means the ribosome now has to find the

start codon and then position it to the

P site. So the 4DS subunit essentially

begins scanning the mRNA for a start

codon and this process is known as the

ribosome scanning which occurs in the

fivep prime to threep prime direction.

This 43S pre-initiation complex now

after binding the mRNA assumes an open

confirmation because the mRNA entry

channel is open because the ribosome now

has to scan the mRNA and this scanning

process to find the AUG consumes energy

in the form of ATP. The open complex of

43S transitions to the next stage when

it finds this AUG.

And let's just draw out the next stage.

This will take me a quick minute.

Let's just tweak this here and some

lines and add some colors

and we're all done. So the change to

notice here is that the AUG is now

paired at the P site with the initiator

tRNA. The EIF4A and 4B remain bound near

the E site and the EIF 4G and 4E move

out of this complex but are still

associated together which allows the

mRNA to be in a loop. Now there could be

many AUGs right? So how do you know

which AUG should be positioned at the P

site? It turns out that this positioning

happens in the context of a sequence

which usually is this G and C-rich

sequence especially in the consensus of

GCC RCC Aug and this sequence interacts

with EIF1 factor and remember that we

said that it helps in the positioning of

the mRNA and that's where it comes into

play and this sequence here is the KAC

sequence and now there's a small catch

in this recognition of mRNA note that

40S S subunit contains the 18S ribosomal

RNA but instead of ribosomal RNA it is

the initiation factor which helps in the

recognition of the KAC sequence. This is

very different from procariats where we

saw that the 16S ribosomal RNA was

involved in the mRNA binding as well as

the positioning of the start codon at

the P site. Here in ukareiots the mRNA

binding and positioning is initiated by

initiation factors. So the 18S ribosomal

RNA here is primarily involved in the

structural configuration of the 40S

subunit. I hope this is clear. All

right, let's move on. So when this

pairing between the mRNA and the tRNA is

established at the P site, the 40S

subunit undergoes a confirmational

change at the mRNA entry channel and it

goes into a compact state and we call

this state a closed state. So this

entire complex moves to a 43S closed complex.

complex.

This complex is also called 48S

initiation complex. Once this complex is

established, the ukarotic initiation

factor 5 causes the GTP in the EIF2 to

break a phosphate bond which then as a

result causes the EIF2 to form a complex

with the GDP. And this complex loses its

affinity towards the ribosome subunit

and as a result it is released. The

EIF-1 and EIF2 interact closely. So

actually both of them get released and

now this causes the Eiff scaffold

protein to release as well. And once the

scaffold protein is out nothing really

stays on the ribosome. So all initiation

factors except for EIF1A which is at the

A site are released. And if we draw out

the resulting picture of the 40S

subunit, you will find that ukareotic

initiation factor 1A is the only one

which is present which is at the A site

and the initiator tRNA with its

methionine is attached at the P site and

the mRNA is still looped as the EIF4E

and 4G are still interacting

independently of the ribosome subunit.

So to make this explicit, the A site is

blocked by ukareotic initiation factor

1A. The P site is blocked by the tRNA

and the E site is empty. Remember that

we said that the EIF-1 prevents the 60S

subunit from joining. Well, now we don't

have the EIF-1 present anymore. So, the

60S subunit can happily bind to the 40S

subunit. But there's a small catch. The

60S subunit needs help. It cannot just

bind by itself. So another initiation

factor EIF5B

which is in complex with the GTP binds

to 60S and delivers it to the 40S

subunit. Specifically the EIF5B

interacts with EIF-1A and correctly

positions the 60S subunit. And when both

of these subunits are correctly

positioned, the GTP in the EIF5B is

reduced to a GDP. And this causes this

5B factor to lose affinity and it gets

kicked out. But when it is being

released, it takes the initiation factor

1A with itself. And now finally we are

left with 40S and 60S subunits that are

still attached together with the

initiator tRNA paired at the start codon

at the P site. And the mRNA is looped

because of this interaction between EIF

4E and 4G. The E site and the A site are

both empty. And this complex is called

the ads initiation complex which is

ready for the next stage that is the

elongation. And this wraps up our

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