#48 Biochemistry Translation II Lecture for Kevin Ahern's BB 451/551


Uploaded by oharow on 06.03.2012

Transcript:
Captioning provided by Disability Access Services
at Oregon State University.
[class murmuring]
Kevin Ahern: Okay folks, let's get started!
Happy Monday.
Nobody's ever happy on Monday.
I am.
Student: I'm happy.
Kevin Ahern: You're happy?
He's happy.
Alright, so we are in the process of translation.
Today we're going to look at it up close and personal,
and at first I'll start out with some very general things
and then we'll move into some more detail.
Everybody I think who's ever taken high school biology
knows about the genetic code
so I'm not going to spend any significant amount of time on it,
but I will point out obviously that the genetic code
is the three-nucleotide designation that specifies amino acids
for making into proteins.
The genetic code is read off of messenger RNAs which are in turn
read off of DNAs, and the genetic code has some features
that are interesting about it.
One feature is redundancy.
There are 64 possible combinations of three-letter codes,
and to read this one would go through, for example,
read this as U, U, and then U,
would correspond to the very top one.
U, U, G would correspond to the bottom one here.
What you notice in looking at the code
is that there is as I say redundancy
meaning that most amino acids
are specified by more than one three-letter designation.
The arrangement of these is not random.
If we look, for example, at C-U-anything, that is CUU, CUC, CUA,
CUG, we see that they all specify leucine.
And what that tells us is that the third position
for most of the genetic code is the least important position.
It's the least important.
It's where we talk about having wobble,
wobble referring to the fact that for many of the codons
the third position can be almost anything,
and if it's not almost anything it is certainly biased
in terms of purines versus pyrimidines.
Look up here at leucine here.
UUA, UUG, okay?
So it's U-U-purine specifies leucine.
Similarly if we look up here at tyrosine,
UAU, UAC, U-A-pyrimidine specifies tyrosine.
So the genetic code is important.
Some amino acids are only specified by one codon.
There's one right there.
Tryptophan is only specified by UGG.
The genetic code also has what are called punctuation features
and the punctuation features
refer to what's called a start codon,
and the start codon as you probably know is AUG,
and AUG specifies methionine.
Methionine is almost always the first amino acid
that is built into proteins
whether it is prokaryotes or eukaryotes.
You'll notice also that that is the only codon
that specifies methionine, AUG.
AUG tells cells where to start making a protein
and we'll see how prokaryotes and eukaryotes
do that a little bit differently with respect to each other
but they both do in fact start with methionine.
The other punctuation marks of the code are the stop codons,
of which there are three.
And yes I think you should know the sequence
of the start codon and the three stop codons.
The three stop codons are UGA as you can see here,
UAG which is seen here-I'm sorry, UAA,
and UAG which is seen here.
So those are the three stop codons.
The genetic code is what we describe as universal
meaning that essentially every cell uses the same code.
There are some very minor exceptions to the code.
There are some mitochondria for example
that use a slightly modified code,
and the slight modifications
usually involve the change of one codon.
So for example some places UGA which is the stop codon here
is also used to code for tryptophan.
So it's not absolute but for our purposes
we will refer to it as universal.
If we compare the genetic code of human beings
to the genetic code of E. coli, a single-cell prokaryote,
they're identical.
They're identical.
So that means that if I take a protein sequence
and I insert it into E. coli, if I set things up properly
I can have E. coli translate my protein coding sequence
and make human proteins in its cells.
That's one of the roots of biotechnology
as we have talked about.
When we think about the pairing that exists
between the messenger RNA and the tRNAs
which is what I'll get to in just a little bit
-I keep jumping to the wrong one.
There we go.
Where did I put that?
There it is, right here.
We can envision that there is a base pairing that has to occur
between the transfer RNA
which brings in the amino acid for translation
and the messenger RNA that's being held by the ribosome.
This schematic shows that base pairing.
And you'll see something different in this.
On the bottom you see the sequence of a messenger RNA.
The messenger RNA is where the codon,
and the codon is the three-base sequence
that specifies an amino acid,
the messenger RNA is where the codon is located.
The complementary sequence to that codon is called the anticodon
and it's contained at the end of a transfer RNA.
Now you see something interesting here.
C pairs with G, G pairs with C,
but in that third position you see a different base.
It's a base that you've seen before.
It's called inosine.
And inosine you may recall was the branch nucleotide
that either specified A or G during nucleotide biosynthesis.
I turns out, the reason I is present there
is I has several stable pairings.
You can see in this case I will pair with C.
Now why do cells do that?
Well they do it for efficiency purposes.
If you recall I said that the third position of the codon,
that is the third position right there where that C is,
is a wobble.
It can vary.
Well if you use a third position up here in the anticodon
that is a base that can pair with multiple things
it means you don't have to make a corresponding transfer RNA
for every single possible third base of that codon.
One or two transfer RNAs will suffice.
Now I will remind you that when we do base pairing
we have to do base pairing antiparallel.
So we see 5' to 3' going left to right on the mRNA
and we see 5' to 3' going right to left in the transfer RNA.
If we look at the pairings that inosine can do
we see the following.
Inosine can pair with U or C or A,
and that means that by putting an inosine
in that wobble position that only one transfer RNA
will handle all three of those bases.
That's a very useful thing.
It saves the cell time and energy
in having to make messenger RNAs.
Another thing that we see with RNA
is that G/U base pairs are stable.
G/T base pairs in DNA are not stable.
G/U base pairs are stable and that means that U can pair
with A or G and G can pair with U or C.
So there's some fluctuation that can happen
with the stability of the pairings between two different RNAs.
That just simply shows the hydrogen bonds
and no you don't need to worry about the hydrogen bonds
and so forth, but suffice it to say that inosine
gives some flexibility with respect to pairings
of the transfer RNA to the messenger RNA.
Well this figure schematically shows
what a transfer RNA looks like.
In fact in three dimensions it doesn't look anything like this.
It's sort of a bent over structure.
But in two dimensions this is the way one would draw it.
You'll notice it has extensive secondary structure
and tertiary structure meaning that it has base pairs
within a given strand.
This is one single strand of RNA.
So this guy is pairing within itself
as we have seen other RNAs do.
And looking at this you can also see
by the lettering that's here
there are quite a few modified bases that are present.
So there's a UH2.
There's a pseudouridine right there.
There's a pseudouridine there.
This is a modified uridine residue here.
There's quite a few modifications.
There's a methyl G, et cetera.
And again you don't need to worry about the specifics of that
but suffice it to say that there's quite a bit
of chemical modification that happens to transfer RNAs.
At the very bottom of the structure you see the anticodon
that I referred to earlier, and the anticodon
is the part that pairs with the messenger RNA.
Up at the other end is where the 3' end
of the messenger RNA is.
That's where that CCA sequence is
that I've talked about before, and that CCA sequence
is the place for attachment of the amino acid.
Now in order for the genetic code to be functional
the proper amino acid has to get put onto here
corresponding to the anticodon
that will pair with the messenger RNA's codon.
So it turns out that there are enzymes
that will correctly read the anticodon
and put the proper amino acid onto the 3' end
of the transfer RNA.
Now I'll say a little about those.
Some people describe that as the first step
in the process of translation.
I will-there's more . . . blah.
This is the actual structure if we try to depict it
in three dimensions and it's a little harder to see
that actual structure that's there.
But that's basically a 3D projection of the overall molecule.
And this is another projection of the same thing.
And up here, way over here
is where this amino acid gets attached.
Well I said that there are enzymes that will in fact
attach the proper amino acid to the proper transfer RNA
with that corresponding anticodon.
That's really critically important
because if random amino acids get put onto transfer RNAs
then the genetic code won't have any meaning.
There has to be a proper linkage between the anticodon sequence
and the amino acid sequence.
Well it turns out that cells have enzymes
called aminoacyl-tRNA synthetases.
There's the magic name right there that you want.
Aminoacyl-tRNA synthetases.
These are enzymes that perform that catalysis.
And it turns out there's one enzyme for each amino acid.
One enzyme for each amino acid.
So there are twenty aminoacyl-tRNA synthetases.
And if there are different tRNAs for that same amino acid
then those synthetases have to also be able to accommodate
slightly different tRNAs
in order to put the proper amino acid onto there.
This shows us, starting from top to down,
this is the far end of that tRNA.
I wish they would use a consistent designation.
Here they put it at the bottom.
But here is the very 3' end of that transfer RNA
that I schematically showed you before with the 3' end
being at the top and we can see that the amino acid
has been covalently attached via an ester bond
to the terminal A residue that's on that tRNA.
So what that enzyme is catalyzing is the formation
of an ester bond of the appropriate amino acid
to the A residue at the end.
Specifically it's getting onto the ribose.
It's not going on the adenine,
it's going onto the ribose of the A nucleotide.
In this case we see that the attachment is to the oxygen
on carbon number three of the ribose.
Some aminoacyl-tRNA synthetases
will attach it to carbon number two.
But you can see that either is available
and that's where this guy gets attached.
One of the things we discover about this attachment
is that this bond right here between the amino acid
and that ribose of the adenosine,
this bond is very very unstable.
Very unstable, meaning that if it encounters water
in about a half a second
half of the molecules will lose this amino acid.
It'll be cleaved.
So that means that once this bond is made
there has to be protection to keep it from interacting
with water, and we'll see that's actually a consideration
in the function of some of the proteins in translation.
I said it was critically important
that the aminoacyl-tRNA synthetases properly read the anticodon
and put the appropriate amino acid on.
And we've seen that cells are very careful
in all of the things that they do.
We saw in DNA replication that there were proofreading,
there was even postreplication repair that happened
with respect to damage or other things in the DNA.
I've talked about how transcription has some built-in controls
to ensure that transcription is fairly accurate.
What you see on the screen
is a depiction of an aminoacyl-tRNA synthetase
in which proofreading of another sort is actually happening.
These enzymes have a way of checking,
"Have I put the proper amino acid onto the tRNA?"
So they don't just put it on,
they actually check to see if they got the right one on there.
This happens as a result of action of an editing site
that's within the aminoacyl-tRNA synthetase.
The amino acid gets put onto one place in the enzyme
and then the structure flips and the editing site
checks to make sure that the right amino acid
has been placed in there.
It's slightly more complicated than that
but for our purposes
that's a proofreading function that's occurring.
Jodie?
Student: And this is while it's still bound
down here at the anticodon?
Kevin Ahern: Still bound to the anticodon, yep.
This is kind of a, I kind of like this figure
although it's kind of big and hairy and complex.
On the right side you can see this aminoacyl-tRNA synthetase
schematically drawn for you.
You can see that one end of it is reading the anticodon loop.
The other end of it is putting on the appropriate amino acid
and then there's the editing site where it flips over
and checks to make sure everything works okay.
It's a very cool and complicated structure that's there.
Now, one of the things we've learned
about analyzing the interaction
between aminoacyl-tRNA synthetase
and the transfer RNA is that the anticodon loop is only part
of what the aminoacyl-tRNA synthetase actually reads.
It turns out that tRNAs differ slightly from one to another
in terms of their internal sequence as you can see here,
and these yellow ends indicate possible places
where aminoacyl-tRNA synthetases can actually read
and confirm that they've gotten the right amino acid
onto that transfer RNA.
So there are other things besides the anticodon loop
that vary from one tRNA to another.
The aminoacyl-tRNA synthetases
is tuned into those differences as well.
Well all of these ensure of course that transitional fidelity,
meaning getting the right amino acid
into a protein is reasonably high.
When we look at the aminoacyl-tRNA synthetases
what we discover is that they fit into two groups,
one group of ten and another group of ten.
And though there are other differences
besides what I'm going to tell you I will just keep it simple
and say that the Class I enzymes have a different mechanism
than the Class II enzymes.
They actually do look at different things on the Trna
and, in addition, they differ
in where they put their amino acid.
Do you remember I pointed out that that ribose,
the amino acid could go on to carbon number two
or carbon number three of ribose.
It turns out that Class I enzymes,
and no you don't need to know which ones are in which class,
but Class I enzymes put the amino acid onto carbon number two.
Class II enzymes put the amino acid onto carbon number three.
This schematically shows,
and again you don't need to worry too much about the details,
the different ways in which the different enzymes
actually bind to the tRNAs.
You can see that the Class I enzymes bind on one side
of the transfer RNA.
The Class II enzymes bind on the 180°
on the opposite side of the transfer RNA.
Well that's some pretty basic stuff.
That first step though, that's called charging an amino acid.
That first step is the attachment of the amino acid to the tRNA.
So when we talk about the process of translation
we think of it as occurring in really four steps,
the first step being the charging of the amino acid,
putting the amino acid onto the proper tRNA.
Once the proper amino acid has been put onto the tRNA
it's important of course for that amino acid
and that bond between the amino acid and the tRNA
to be protected from water, and as I said
we'll see some proteins involved in that process.
The actual translation of a sequence is performed
as you know by ribosomes,
and ribosomes are really interesting oddball structures.
Ribosomes contain what are called two subunits,
a large subunit and a small subunit.
And each subunit has multiple proteins.
There's about fifty proteins in a ribosome.
Many many proteins involved in this bigger structure.
But as you can see here, the colors that jump out at you,
the things that jump out at you are actually the ribosomal RNAs.
A ribosome contains not only proteins
but ribosomal RNA molecules.
Before I say about those
I probably should talk about this designation: 30S, 50S, 70S.
So the way people measure sizes of macromolecular complexes
is by the rate with which they get forced
by centrifugal force through a medium.
The faster it moves, the different the value that it has.
The smaller a molecule is,
the smaller a molecule is the different will be the rate
as it passes through a solution.
So a smaller guy is not going to move as fast as a bigger guy
is going to move through if everything else is equal.
And that rate with which things move is called a Svedberg unit,
S-V-E-D-B-E-R-G.
That's what this S refers to here.
So the small subunit of a ribosome in E. coli has a size of 30S,
thirty Svedberg units.
The large subunit moves faster.
It has a size of fifty Svedberg units.
And if we put the two together as an attached ribosome
you'll see that the Svedberg units are not additive
but they make a larger 70S.
So this is the intact.
These two don't obviously add up to seventy.
Yes?
Student: Is sedimentation coefficient also acceptable?
Kevin Ahern: Yes, sedimentation coefficient is also acceptable
if you want to call it that instead of a Svedberg unit,
that's correct.
These sizes are different in eukaryotic cells
but they're the same relative amount.
So in a eukaryotic cell the small subunit has a size of 40S,
the large subunit has a size of 60S,
and the combined ribosome has a size of 80S.
So if I talk about a 80S ribosome
I'm talking about a eukaryotic ribosome.
If I'm talking about a 70S ribosome
I'm talking about a prokaryotic.
Now the sizes really aren't the most important thing here.
The ribosomal RNAs play very important roles in this process.
If we look in E. coli, which is the simpler system,
there are three ribosomal RNAs.
Three ribosomal RNAs.
The 30S subunit contains one of those.
It's called the 16S.
And again, that's a Svedberg unit again.
The 16S ribosomal RNA.
The 50S subunit, the larger one contains two.
It contains what's called the 18S
and-I'm sorry it contains the-
actually I'm getting my numbers wrong now.
16 and yeah, I think it's 23S.
23S is the larger ribosomal RNA that's in E. coli.
The 23S and a smaller one called a 5S
are both contained in the large subunit.
Now each of these has different functions.
I'm going to give you functions for the 16S
and for the 23S as we get going along.
And I haven't said those functions yet
but I will save that for a second.
When we go to translate a message, and I'm talking here not "we"
but E. coli, when E. coli goes to translate a message,
as I said, all cells start with methionine.
In prokaryotic cells, however, they start
with a modified form of methionine.
So their initiator, what's called their initiator tRNA,
that is the one that's going to come in
and bring in that very first amino acid,
brings in not methionine but something called formylmethionine.
Formylmethionine looks like this,
and no you don't need to know the structure of that
but it is a chemically modified form of methionine.
It only occurs for the very first amino acid
in a protein in prokaryotes.
Eukaryotes don't do this.
Why is this here?
Well it turns out that in getting
the synthesis of a protein started this very first amino group
turns out to be reactive and it can cause a reaction to occur
that will stop translation if it's not covered up.
So prokaryotes avoid that problem by covering it up
with this formyl group and so the very first amino acid
that's put into a prokaryotic protein is formylmethionine.
We compared sequences of the DNAs where there were genes
and we saw that there were common features
that were located very close to the translational start site.
We called those common sequences
TATA box as part of a promoter, for example.
If we do the same thing for messenger RNA sequences
and we compare them to our location relative to the start site,
the start being AUG-in some cases you see alternatives used.
There's a GUG right there.
It's not common but it does happen occasionally.
If we compare those sequences we see a smattering
of conserved sequences there kind of like what we saw
with the promoter.
These conserved sequences are within ten base pairs of the-
or not base pairs, but ten bases of the start site,
this being number one.
There's minus one, two, three, four,
five, six, seven, eight right there, okay?
The consensus of this sequence is GGAGG
and it turns out this sequence
has a very very important function.
First of all it's called a Shine-Dalgarno sequence,
S-H-I-N-E dash D-A-L-G-A-R-N-O.
It's named for the people who discovered it.
The Shine-Dalgarno sequence
is a sequence that can form base pairs
with a region of the 16S ribosomal RNA in the small subunit.
Well why is that important?
The reason that that is important is it is telling
the cell where to start translating.
Where to start translating.
That turns out to be important because as we can see
there are many places where there are other-
there's an AUG right there, okay?
This helps to position the proper AUG
in the place where translation is going to occur.
So the function of the Shine-Dalgarno
is to tell the ribosome where you start translation
because that proper AUG is placed into the start place
for translation to occur.
The Shine-Dalgarno sequence is found in prokaryotic cells.
It is not found in eukaryotic cells.
Eukaryotes use a different approach.
When I showed the schematic figure of the ribosome
the other day I pointed out that there were three sites
that we were concerned about.
I called them E and P and A.
If we look at this ribosome here, there is the A site,
there is the P site, there is the E site.
And they are specific locations where a codon can sit.
A specific location where a codon can sit.
In the process of translation we can imagine
that there is a messenger RNA running through this ribosome.
Running through the ribosome right here.
Translation, like every other process you've seen
occurs in the 5' to 3' direction.
So the 5' end of the ribosome would be over here.
I'm sorry, 5' end of the messenger RNA would be over here.
The 3' end of the messenger RNA would be over here.
And the ribosome would be moving from left to right
in translating that sequence.
That means that the incoming codons
where the transfer RNAs will pair,
the incoming codons will first appear in the A site.
In the process of translation the ribosome
will move along the messenger RNA.
They will then move to the E site-I'm sorry, to the P site
and then finally to the E site and then they exit.
And E stands for "exit."
So translation is occurring 5' to 3' one codon at a time,
one three-base sequence at a time,
and these sites provide places for one codon to sit.
This now schematically shows what is happening
in the process of translation.
This is actually jumping the gun just a little bit
because the ribosome has already been assembled
and we're going to say a little bit about that,
but after the ribosome has been assembled
we can see what I just told you in words.
That is, there's the messenger RNA 5' end on the left side,
3' end on the right side.
There's the A, the P, and the E.
And not only can we see in this schematic the messenger RNA
but we can also see the anticodon loop
of the transfer RNAs holding amino acids.
What we see here is a protein sequence
that's in the process of being made.
This already has two amino acids and in the next step
this green guy is going to get attached to this purple guy.
As I say this jumps the gun a little bit
but it gives you an overview
of what's happening in this process.
Okay I'm kind of going kind of fast so I'll slow down
and ask if you have any questions
before I dive into some mechanism.
Yes sir?
Student: A little bit on the representation
on those you show the two dimensional as sort of a line
with your amino acid at the bottom, down at the bottom.
But in reality, since those are L-shaped
do they kind of go in and out of the plain
or are they sideways in the . . .
Kevin Ahern: Yeah that's a good question.
So the question is tRNAs aren't flat
and I'm drawing everything in flat structures.
Yes there's a very important
three dimensional component to all of this.
So these are all very schematic drawings.
Maybe we'll take a break and do a song.
How about that?
I've got several songs about translation
so we'll do one of them here.
The Codon Song.
[singing The Codon Song]
Lyrics: Building of proteins, you oughta know
Needs amino A's
Peptide bond catalysis in ribosomes
Triplet bases, three-letter codes.
Mixing and matching nucleotides
Who is keeping score?
Here is the low down
If you count codons
You'll get sixty-four.
Duh duh duh duh duh-duh duh-duh duh duh duh!
Got to line up right
16S RNA
and Shine-Dalgarno site
You can make peptides, every size
With the proper code
Start codons positioned
In the P site place
Initiator tRNAs
UGA stops and AUGs go
Who could ask for more?
You know the low down
Count up the codons
There are sixty-four.
[stops singing]
Nobody knows that song except me, I think.
[laughter]
And I don't know it very well.
Well we're at the second step in that translation process,
the first step being the charging of the amino acids
by the aminoacyl-tRNA synthetases.
That's happening away from the ribosome.
Everything we're going to talk about now occurs in the ribosome.
The very first step in that process is initiation.
And as I mentioned with respect to comparison to transcription,
initiation, elongation, and termination
also are three phases of the translation process.
What you see on the screen is a depiction
of the initiation phase and the initiation phase requires
that this ribosomal complex be assembled.
This actually has to assemble before translation can occur.
The assembly requires several things.
First, it requires the small subunit to start.
It requires three initiation factors.
And they've very simple.
They're called IF1, IF2, IF3.
We're not going to distinguish their functions.
You'll also see when we get to the elongation phase
that there are three elongation factors as well.
The third thing that we need to get everything started
is a messenger RNA to translate.
The initiation factors facilitate a couple of things.
One is the binding of the messenger RNA to the small subunit,
and the second is bringing in the very first initiator tRNA.
That's the formylmethionine tRNA.
This is the only tRNA that gets its start in the P site.
It actually starts in the P site.
And you'll notice that this is called IF2.
That is what brings in that formylmethionine.
That formylmethionine is brought in by IF2,
and yes, IF2 is protecting that bond against water.
So it's covering up the bond.
It's not showing it very clearly here
but it's covering up the bond between the tRNA
and that formylmethionine so that water doesn't cleave it.
After these things have been positioned properly,
and the other thing that has to happen here
is the Shine-Dalgarno sequence in the messenger RNA
has to be aligned with the 16S sequence
so that we know which is the proper AUG
to put into the start site which is the P site.
After this has been aligned properly
then we can add the large subunit and everything is,
the initiation phase is done at that point.
So the initiation phase basically involves
assembling the ribosomal complex, three initiation factors.
The one I told you the function of is IF2
which brings in the formylmethionine, puts it in the P site,
and we're ready to start.
So you'll notice we haven't translated anything yet.
All we have at this point is a ribosome
with an aminoacyl-tRNA formylmethionine sitting in the P site.
That's all we have.
We have what's called the 70S initiation complex at that point.
Well elongation is where the peptide bonds
are actually formed and that occurs here.
So here's where we finish initiation.
There's the P site.
And we can see that there's the messenger RNA
that it's paired to and up here in the 50S subunit
is the other part of the P site
which is where the amino acid is sitting.
Another amino acid, aminoacyl-tRNA comes in.
And there's two possibilities.
One is that this anticodon is perfectly paired
in which case the right aminoacyl-tRNA has come in,
or it's not going to properly pair
in which case the wrong one has come in.
You'll notice I'm calling it an aminoacyl-tRNA.
I'm distinguishing that from an aminoacyl-tRNA synthetase
which is the enzyme that puts it on.
We call the complex an aminoacyl-tRNA.
Now, this incoming tRNA is not bare as it shows here
but in fact it's carried in
by something called an elongation factor.
There are three elongation factors
and we're going to talk about two of them.
The one that carries in the aminoacyl-tRNA has to, yes,
protect this bond against water.
And in E. coli this elongation factor is called EF-Tu
which sounds very much like IF number two, doesn't it?
EF-Tu.
EF-Tu covers this guy up, it carries it in,
and it does a couple of other things.
EF-Tu is something we call a G protein.
What do G proteins do?
Well G proteins you may recall from last term
are involved in carrying GTP and they can cleave it for energy.
Well in fact that's what happens here.
If the proper one is inserted, that is this base pairing
is properly in the A site, the EF-Tu says,
"Okay, my job is done."
It cleaves its GTP and it gets out of there.
That leaves the proper amino acid
in the ribosome protected from water
because the inner part of the ribosome
is also protected from water.
If the wrong aminoacyl-tRNA synthetase has come in-
I'm sorry, aminoacyl-tRNA has come in
then these base pairs won't be proper
and EF-Tu will pull it out and leave.
So this is one of the ways in which we ensure
that the proper aminoacyl-tRNA is in the A site.
That's thanks to the work of EF-Tu.
EF-Tu as you might imagine
is a very important protein in E. coli.
It is in fact the most abundant protein in E. coli.
That sort of makes sense
because you've got to have plenty of it around
once you've charged a tRNA onto, with an amino acid,
you'd better have something there to cover up that bond
pretty quickly or you're not going to have
the product that you want.
So we've brought this in.
We've got one aminoacyl-tRNA in the P site,
one aminoacyl-tRNA in the A site.
Each is attached to their own amino acid.
They haven't been joined yet.
That happens in the next step.
The next step is there's a peptide bond
which joins these two guys together.
The joining schematically actually occurs
in this little orange region up here.
The joining is really interesting.
The joining is catalyzed not by an enzyme.
I want to repeat that.
It's not catalyzed by an enzyme.
Peptide bonds are not formed in the cell by enzymes.
They're formed by ribozymes
meaning that there's a catalytic RNA in that ribosome.
A catalytic RNA is an RNA that can catalyze a bond.
And the RNA that's in this ribosome
that's catalyzing the formation of this bond
is the 23S ribosomal RNA.
The 23S ribosomal RNA is a ribozyme, R-I-B-O-Z-Y-M-E.
All the protein on earth is made
thanks to the catalytic action of a ribosomal RNA.
That's true in eukaryotes as well.
Pretty cool stuff.
Well at this point we have a dipeptide.
We see that this guy on the left
is soon going to be on its way out.
It gets kicked out by the process of elongation.
We've already done the first steps of elongation.
Now we have to translocate
meaning we've got to move the ribosome down three more bases.
So notice where we are here.
Notice where we are here.
We've shifted the bottom, we haven't shifted the top.
This involves translocation and this translocation process
is moving these guys further along.
Here we were in A, P, E.
Now we're going to shift this guy.
It's going to move to the left one more.
We're moving to the left.
So now we see the green guy is underneath the orange one
whereas the blue guy was underneath the orange one over here.
We have moved this messenger RNA to the left,
or if you want to think about it
we've moved the ribosome to the right.
Either way, either is equivalent.
Notice now this guy has two amino acids on it
joined by a peptide bond.
This guy has nothing on it.
It's ready to exit and that's why it's in the E site,
because E stands for "exit."
This factor that catalyzes this movement of the ribosome
down the messenger RNA is called EF-G
which stands for elongation factor G.
Elongation factor G is also a G protein.
It also uses GTP for energy and as a consequence
we've moved down, we're ready now, we've got an empty A site
and now we can bring in another aminoacyl-tRNA
and continue the process.
This figure reminds us of something very important.
All of the energy in translation...
All of the energy in translation comes from GTP, not ATP.
All the energy of translation comes from GTP.
There's a third elongation factor.
It's not shown on the screen
and I'm not going to talk about it.
But suffice it to say there is a third one
that's important as well.
This is usually a little confusing so I'll stop
and take questions on this.
Yes, Jerry?
Student: It looks like there's an EPA site also on the 30S?
Kevin Ahern: There is, yes.
Student: For translocation, are these essentially
kind of like an active site that pulls off a tRNA.
And that translocation, does that pick out that 30S tRNA?
[Inaudible] dragging force that moves the tRNA away from...
Kevin Ahern: So the only movement that's happening
is the actual, and these ribosomes
are sort of shuffling with respect to each other.
That's the moving that's happening
and as a result of that shuffling
this is sliding down the messenger RNA.
Yeah, so there is, and you can see here,
like the top is slightly off
compared to where it was right here.
Student: But that lower 30S blue site [inaudible]
pulls the tRNA off of the mRNA?
Kevin Ahern: You're talking about right here?
Student: Yes, right there.
Kevin Ahern: So I would not call this an active site, no.
Active site I would use solely
for the purpose of describing catalysis.
So as you could imagine this is geometrically different
so it's favoring the breaking of the hydrogen bonds
between these guys right here.
And remember we've only got three nucleotides there
so there's not a lot of strong things
that's holding that together.
Yes, Jodie?
Student: I've heard this described previously
as ratcheting but in that particular course
the instructor said that your E site remained filled
until the next one bumped it out.
They all three remained filled and in association.
Is that accurate?
Kevin Ahern: It's not my understanding that that's accurate.
It's my understanding that the E site
actually doesn't stay filled for very long, no.
I'm not an expert in this though, I will tell you that.
So that's a pretty cool process.
We've gone through initiation, we've gone through elongation.
There's translocation which we don't need
to worry too much about.
I think it confuses the picture a little bit.
EF-G looks kind of like a transfer RNA
and so it gets into the A site as you can see there
and kind of pushes things along.
Here's EF-Tu.
EF-Tu as I said is the most abundant-
actually that's not EF-Tu at all.
Alright, my link is bad.
This is showing the process of termination.
So that elongation process will go on and on and on
until a stop codon appears in the A site.
When a stop codon appears in the A site
there's nothing that will compare with it
because there's no tRNAs that are complementary
to the stop codons in the A site.
So this guy will sit here for awhile.
And finally something called a release factor will come in
and the release factor does one thing.
It carries, first of all it goes into the A site.
It forms a base pair with the UAA.
And yes, it is a protein.
What it does is it carries into the inner workings
of the ribosome a molecule of water.
And guess what the water does?
It cleaves the bond between the amino acid
at the terminus of the polypeptide chain
and the last tRNA it's attached to.
That molecule of water favors the release.
Now you can see the polypeptide has been released
by the ribosome and everybody is home free.
I didn't mention but I'll mention briefly here
that you see that this is exiting out the top
of this large subunit.
There's actually a little tunnel
through which the growing polypeptide chain
emerges as translation occurs.
That's a good stopping point for today.
Let's call it a day and I will see you on Wednesday.
[class murmuring]
[END]