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.
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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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