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ELIZABETH NOLAN: Today we should be completing the translation

cycle.

And the next topic that will come up

is use of antibiotics as tools to study the ribosome

and translation.

So just a recap from last time, we went over the delivery

of aminoacyl--tRNAs by EF-TU and looked at this model

for understanding how that happens, OK?

So recall, we discussed the initial binding

of the ternary complex of EF-TU GTP and the aminoacyl-tRNA.

And that's codon independent.

When a codon/anticodon match occurs,

we have a push in the forward direction.

EF-TU's a GTPase.

There's activation of the GTP center.

So conformational changes, GTP hydrolysis.

So we have EF-TU and the GTP bound form.

There's conformational change, and ultimately

accommodation of this tRNA in the A site.

And that allows for peptide bond formation.

So where we left off last time was

discussing the conformational changes

that occur in the decoding center

and also in the GTP center of EF-TU.

And just to highlight, I mentioned

that there are conformational changes within the 16S rRNA,

in particular three nucleotides that

occur when it's a cognate codon/anticodon interaction.

And these are just shown here.

And effectively what we're looking at in these three

panels are the 16S rRNA in the absence of the tRNA,

in the presence of the tRNA, but the tRNA

is removed from this image for simplicity,

and then with the tRNA bound.

So some of the easiest changes to see here

are with A1492 and A1493.

So if we look in the absence of tRNA,

they're pointing down the bases.

And here as a result of tRNA binding in the A site,

we see that A1492 and 1493 are flipped, flipped up.

OK.

And if we look here, you can see how these are interacting

with the bound tRNA.

OK.

So this conformational change helps

to accelerate the forward steps.

So that's in the decoding center.

And then also just remember 70 angstroms away

in the GTP center of EF-TU, there's

conformational change of these hydrophobic residues that

are thought to be a hydrophobic gate that allows histamine

84 to activate a water molecule for attack in GTP hydrolysis.

So at this stage we're finally ready to have a peptide bond

formed by the ribosome.

And so we need to think about that mechanism and then

what happens after.

I'll say, so effectively what we have in the P site

is the tRNA with some growing peptide chain.

[WRITING ON BOARD]

And then we have the aminoacyl-tRNA in the A site.

[WRITING ON BOARD]

And so what happens effectively, we have attack from here,

release such that we end up with a P site

with a deacylated tRNA.

And in the A site we now have the peptidyl-tRNA that

has grown by one amino acid monomer.

[WRITING ON BOARD]

Here, OK?

OK.

And so this is the N- terminal end

of the protein or the polypeptide,

and here's the C terminal end here.

So thinking about this mechanism and having nucleophilic attack

from this alpha amino group of the aminoacyl-tRNA in the A

site, what do we need to think about?

Is there anything surprising or unusual?

AUDIENCE: Think about protonation state.

ELIZABETH NOLAN: Yeah, right.

Exactly.

We need to think about the pKa.

So typically do we think about an alpha amino group being

protonated or deprotonated, that physiological pH.

Yeah.

Protonated we typically think about an H3+, not an H2 here.

So what does that tell us?

There has to be a general base somewhere

that deprotonates this alpha amino group,

such that we have this species that can attack,

and then can imagine just formation and collapse

of a tetrahedral intermediate here.

So what is the mechanism of catalysis?

OK.

Our room's possessed.

So what is the mechanism of catalysis here?

What do we know?

So we know from looking at the structure

that the ribosome is a ribozyme.

So no proteins in the catalytic center.

What else do we know?

There's no metal ions there and there's no covalent catalysis.

So really what is a paradigm here?

We have a paradigm of conformational change

and effectively we have substrate positioning.

You can imagine there's some protons shuttling

in an electrostatic network that allows this to happen.

And so as soon as this aminoacyl-tRNA

enters the A site, we have formulation

of this peptide bond.

So what needs to happen next-- and once the screen gets fixed,

we'll look at an actual depiction

of these players in the PTC.

What needs to happen after the peptide bond forms--

and we have now this peptidyl tRNA in the A site

is that before the next round of elongation effectively

we need to reset, and the mRNA and the tRNAs

need to move relative to the ribosome.

OK.

So effectively we need to get this deacylated tRNA to the E

site, and we need to get this peptidyl tRNA to the P site

such that the A site is empty.

Is it not going down?

Pardon?

OK, that's fine.

So this process is called translocation here,

and effectively we can just consider the three sites.

We have the E site, the P site, and the A site.

[WRITING ON BOARD]

OK.

And in this process another elongation factor,

this time elongation factor G is involved.

And the outcome is that we end up

with the deacylated tRNA in the E site,

the peptidyl tRNA in the P site.

OK.

And then the A site is empty such

that the next aminoacyl-tRNA can come in.

OK.

So immediately after peptide bond formation, and this

is the state after the process called translocation.

So EFG is also a GTPase here.

And effectively what happens is that EFG

bound to GTP binds near the A site and GTP hydrolysis occurs.

Bless you.

OK.

And as a result of GTP hydrolysis,

there's conformational change.

OK.

And this results in translocation and then EFG

is released.

And in thinking about translocation

we think about two steps.

OK.

And so the first step is something called formation

of hybrid states, and then the second step

is the actual movement, the mRNA and tRNA

relative to the ribosome here.

So we'll take a look at this.

So here's just an overview of peptide bond formation

backtracking a little bit, and then back to here.

Thinking about confirmations and what

this looks like, what we're seeing here in this depiction

is the P site tRNA in green, we have the A site tRNA

in this red color, and then we see the 23S rRNA shaded

in light blue in the back.

OK.

So here's A76, here is an attached amino acid,

and we see the nucleophile here, and attack there.

OK.

So substrate positioning within this active site.

Here, as I talked about this translocation process,

we're now at this stage as shown with a depiction

of the ribosome.

So this is where we're going using EFG in complex with GTP

to allow the translocation process to occur.

Here is another cartoon depiction.

And so if we begin after peptide bond formation,

we see the incoming EFG in complex with GTP.

And if we look in this cartoon, it

shows EFG binding near the A site.

So it's not binding exactly in the A site, but nearby.

OK.

There's GTP hydrolysis.

We now have EFG in the GTP bound form.

OK.

And now we see translocation, and that these tRNAs

have moved.

And after this step, EFG is released.

And at this stage the ribosome is

ready for its next round of the translation cycle.

So let's take a look at what we know

about the structure of EFG.

So this is a really beautiful example of molecular mimicry,

and EFG is shown here.

And if we compare EFG to the complex of EF-TU

with tRNA like the ternary complex shown here, what we see

is they look very similar, and this domain IV of EFG

resembles the tRNA quite well.

So just looking at that we can begin

to think maybe its domain IV that's

coming in near that A site to have some interactions

and cause this translocation event.

Here is just a comparison of the ribosomes with either EF-TU

and tRNA bound, which we've seen before,

and here we see EFG in this red color.

So quite similar, but are they the same?

And the answer is no.

So quite recently a crystal structure

was obtained with EFG bound to the ribosome,

and it was determined that in this structure

EFG is bound in the post translational state.

So what do we see?

If we take a look here, we have a tRNA

in the E site, a tRNA in the P site,

and here in red we see EFG.

And if you take a look, recall that ribosomal protein

L12 that was involved in recruiting the ternary complex,

we're seeing that there's some interaction here with EFG

in that protein as well.

If we look in this panel B--

so we look at a close up view near the A site,

what's happening?

Here's a P site tRNA, here is the mRNA,

and here is EFG bound, and it's domain IV

that's sticking its way in.

OK.

And so what are we seeing here?

Basically these tRNAs have moved at this stage

and we still have EFG bound here.

So is EFG interacting with the A site codon of the mRNA

based on this view?

AUDIENCE: Not really.

ELIZABETH NOLAN: Yeah.

No.

So not really.

So it's not interacting in the same way as the tRNA.

That's the take home here.

They look the same, but the details are different here.

OK.

So what about these hybrid states?

They have two different views, and the slides

for looking at this--

effectively what the hybrid states are--

OK.

These describe basically the orientation of the tRNAs.

So effectively they can be P/E or A/P here.

And the P/E state talks about having the anticodon end in P

and the deacylated three prime end in E. OK.

So here we're referring to this tRNA

that ultimately needs to get ejected from the ribosome.

And A/P we have the anticodon end in A