This video details the termination step of eukaryotic translation, focusing on the roles of termination factors, the mechanism of polypeptide release, and subsequent ribosome recycling, contrasting it with prokaryotic processes.
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in this video we will discuss the termination step of eukaryotic translation specifically
we will discuss the termination factors and focus on one special factor in much more detail
and as we discuss the mechanism of termination we will contrast the process of eukaryotic
termination with the process of prokaryotic translation termination the major difference
that we will see in the eukaryotic termination is that it does not include a translocation step
and we will see why that is the case and ribosome recycling in eukaryotes is also very different
from the prokaryotes and we will discuss that as well the link to the prokaryotic termination video
is down in the description if you need a refresher and hopefully you understand the prokaryotic
translation because it'll make this video very easy to understand so we begin where we left off
in the elongation video where the ats ribosome complex has the trna attached at the p site and
this trna is attached to the polypeptide chain which is protruding into the protein exit channel
in this ats ribosome complex the e site and the a site are both empty or unoccupied you can check
out the eukaryotic translation elongation video if you're not unsure how this ats ribosome complex
is formed the link to that video is also down in the description so the termination starts when a
stop codon is encountered at the a site as we have seen this before there are three stop codons uaa
uag and uga and this ads ribosome complex where the stop codon is sitting at the a site is called
the pre-termination complex before we dive into the process of termination let's talk about the
key players of the mechanism which are the termination factors in prokaryotes we saw
that there are two class 1 termination factors rf1 and rf2 and 1 class 2 factor which is named
rf3 and this helps in the proper functioning of class 1 factors the class 1 factors have
the job of recognizing stop codons and then we also saw ribosome recycling factor
and the elongation factor g which enables a translocation step in the termination of ribosome
and this leads to the ribosome disassembly on the other hand if we talk about eukaryotes we have
one release factor which is called erf1 and this factor alone recognizes all three stop codons
and similar to prokaryotes where rf3 helps the class 1 factors the eukaryotic rf3 also helps
the rf1 and this rf3 is also a gtpase and we will see that the rf3 helps in the polypeptide release
and the positioning of the release factor 1 at the peptidal transference reaction center
at the p site then finally we have a protein complex named abc e1 which simply stands for
atp binding cassette e complex one sometimes people also call this guy rli which stands
for rnase l inhibitor these names are not that important except to keep in mind that the abc e1
generates energy for the ats ribosome splitting and this happens when the abc e1 interacts
with erf1 and after the ats ribosome is split into subunits we will see how initiation factors 2d
1a and others help in removing the trna and the mrna from the 40S subunit there are two
potential pathways for this last step and we will talk about both of them when we get there
the main player in this entire termination process is the release factor one and let's just look at
the structure of this release factor which will make it easier to understand the entire mechanism
because release factor 1 sticks around until the end of the termination process
so this release factor sort of looks like a fidget spinner it has the n-terminal region
which loops out another middle region and then it finally has a c terminal end the c terminal end
is called domain c where c stands for the c terminal the n terminal is called domain n where
n stands for n terminal and this looped out middle part is called domain m where the m
stands for middle the middle domain has the ggq motif that we have already discussed in the
prokaryotic termination as well and this has the job of cleaving the polypeptide from the p site
trna domain n has the peptide anticodon which we also talked about in prokaryotic termination but
in this domain n the motifs responsible for stop codon recognition are different from prokaryotes
they are tsk and tasnix and some others but it's this tas niks that you need to keep in mind
the domain c has a bunch of binding site that are responsible for binding to release factor 3 and
the abc e1 complex just one last thing to contrast here the tasnix motif is very different from the
stop codon recognition motifs in prokaryotes where we saw pvt pat spf and spv motif all right
so this fidget spinner looking like release factor essentially mimics the trna structure and because
it can recognize the stop codons it is brought into this pre-termination complex to bind the stop
codon at the a site but release factor 1 usually comes in a complex with its enhancer protein
which is the release factor 3 which also happens to be a gtpase so it carries a gtp with itself
the release factor 3 binds at the c domain of the release factor 1 but the catch here is that the
domain m which has the ggq motif is actually bent towards the release factor 3. there are
some secondary structures like switch elements and loops and release factors that help in the bending
of the domain m and this ternary complex of release factors is ready to bind at the stop
codon in the ats pre-termination ribosome complex let's just quickly sketch out the structure here
and let's for simplicity just say that the stop codon here is uaa and it is present at the a site
the ternary complex is then positioned in such a way that the domain n of the erf1
contacts and essentially recognizes the stop codon the release factor 3 then positions itself near
the junction of the 40S and the 60s subunits and part of it also touches the factor binding region
so for now in this complex the domain n is positioned where it should be but the domain m
which has this ggq motif is sort of away from the trna on the p site and this distance
is about 80 angstroms so the ggq motif cannot cleave the polypeptide just yet
so after this ternary complex is bound the srl region in the factor binding region
activates the gtpase of release factor 3 which releases the phosphate bond now this termination
factor bound ribosome moves to the next step so following this bond breaking the release factor 1
is properly accommodated by the release factor 3. this simply means that the release factor 1
pairing with the stop codon becomes irreversible so it is now difficult for the release factor 1 to
dissociate away from the a site and additionally as a result of this gtp reaction the domain m
now is moved into the peptide transferase reaction center at the p site and now it is
very closely sitting to the trna and this makes it attack the three prime end of the transfer rna
mechanistically speaking the ggq motif increases the ester bond accessibility for a nucleophilic
attack and when this nucleophilic attack happens the polypeptide is released from the ribosome
which is later folded into a functional protein and when this happens the rf3 gdp complex is also
released the exact mechanism for this release of release factor 3 is unclear but it's partly
because the gdp complex makes the release factor 3 unstable on the ribosome and now we can sketch out
the resulting ads ribosome and you notice that the psi trna has no polypeptide attached to it
which means that the protein exit channel is free and because the release factor 3 is gone
the release factor 1 is alone at the a-site still paired with the stop codon and this ats ribosome
complex where polypeptide is released from the ribosome is called the post-termination complex
and because there is no polypeptide at the trna here the translation process or protein synthesis
technically speaking is finished at this step so which means the only thing that
is left to do for a cell now is to split the ats ribosome into subunits
and now we enter the process of ribosome recycling to start the recycling process the complex abce1
also known as rli enters and this complex has this hinge element which is essentially a helix-loop
helix structure another thing to note about the abc e1 are these two pockets which have
the potential to close but they're open for now so this abc e1 complex is recruited onto this
post-termination complex specifically the hinge element interacts with release factor 1
at the domain c just like release factor 3 and binds at the junction of 40s and 60s subunits
and this abc e1 complex has these pockets in an open form and these pockets close when atp is
bound but when atp is not bound they are open so we call this abce1 configuration
an open configuration and now this ribosome is ready to move to the next step so let's just
draw out the ribosome structure and the transition happens when two atp molecules bind in the pocket
of the abc e1 and when they bind it closes the abc e1 complex and this complex is referred to
as the closed configuration of abc e1 this closed state of abc e1 then triggers a feedback loop
which goes through erf1 and this causes the abc e1 to break the phosphate bond present in the atp and
this phosphate cleavage releases energy and this release of energy from atp is used to split the
ats ribosome into 60s and 40S subunits essentially the hinge element acts like a can opener and
splits the ats ribosome which means that the 6ds subunit is released and this causes the release
factor 1 to be kicked out as well because it is attached with the abc e1 and this atp reaction
causes the abc1 complex to go back to its open complex state but after this subunit splitting the
40S subunit is still attached to the trna and the mrna but both e and a sites are now empty so we
somehow need to release the transfer rna and the messenger rna from the 40S subunit there are two
mechanisms to dissociate the 40S subunit the first mechanism involves initiation factors 3 1 and 1a
which remove the p-site trna and we have seen these guys before in the initiation video the
initiation factor 1 binds the transition of e and p site and the initiation factor 1a binds
the a site and this kicks out the trna from the p site so now the p site is empty and then the eif3
which is this giant initiation factor helps in the release of mrna from the 40S subunit
specifically by interacting through its subunit j which binds at the mrna entry channel this exact
complex where the 40S subunit is attached with these initiation factor is a complex that we also
saw in the initiation step of the translation as well so i hope that the translation process
is now coming to a full circle of understanding so that's one way of recycling the 40S subunit
the other mechanism relies on a different initiation factor initiation factor 2d which
binds at the p site of the 40S subunit and this destabilizes the codon anticodon pairing of the
mrna and trna which is happening at the p site and as a result it causes the mrna and trna to release
and this 40S subunit is now recycled or it could be turned over or it can go into another round of
translation one last note this initiation factor 2d sometimes is also known as ligatin so if you
see this term being used instead of if2d don't be confused they refer to the same factor
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