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ELIZABETH NOLAN: We're going to continue

where we left off last time.

So briefly I'll make a few points about initiation

of translation in prokaryotes.

And then where we're going to spend the bulk of the time

today is with a review of tRNAs and then discussing

the aminoacyl-tRNA synthetases, which

are the enzymes responsible for loading

amino acids onto the three prime end of the tRNA.

And these points are important because these process has

to happen in order for the amino acids

to be delivered to the ribosome, which

is where we'll go on Wednesday.

So the first questions are, how does initiation happen?

So how does this ribosome, 70S ribosome,

get assembled with the mRNA and initiator tRNA bound?

And then we're going to ask, how do we

get an aminoacyl-tRNA, such that the amino acids can

be delivered to the ribosome?

So first, for initiation in prokaryotes,

there's a few steps to this process.

We'll just look at these at a basically superficial level

of detail.

But recall that there are translation factors.

And during initiation, there are three initiation factors--

so IF 1, 2, and 3--

that are required to help assemble the 70S ribosome here.

So first in terms of initiation, what happens

is that the mRNA needs to bind to the 16S RNA of the 30S

subunit.

And so I point this out because at this stage in the process,

the 70S ribosome isn't assembled yet.

So we have the mRNA binding to the small subunit.

And this process requires initiation factor 3.

And effectively what happens is that the mRNA has

a region called the Shine-Dalgarno sequence

in prokaryotes, which is the site of ribosome binding.

And then upstream of that is a start codon

that signals for the start of translation.

So if we think about the mRNA of the five prime end,

and somewhere there's a sequence that

signals for ribosome binding.

OK, and then we have our start codon

that signals the start of translation.

OK.

And so this gets translated here.

OK, so this start codon pairs with initiator tRNA.

And this initiator tRNA is special.

One reason why it's special is because the amino acid attached

is an N-Formylmethionine OK.

So sometimes the initiator tRNA is called f-met tRNA f-met

as an abbreviation there.

So just as some overview here, what

we're seeing in this alignment is

a number of the ribosome binding sites,

or Shine-Dalgarno sequences in prokaryotes.

We have the start codon on that pairs with the initiator tRNA.

And here's a schematic depiction of what I've indicated here

on the board.

OK, so the mRNA binds to the 16S of the 30S subunit.

So the 70S is not assembled at this stage.

And IF3 is involved, as I said.

The Shine-Dalgarno sequence determines the start site.

And we determine the reading frame, as well.

So here is just an indicating translation of a polypeptide.

What happens after that?

So after that, it's necessary to assemble the 70S ribosome,

have the initiator tRNA in the P site,

and have the cell ready to go for translation.

And here's just one cartoon overview

that we'll use as a description of this process.

OK.

So what do we see?

We've talked about this step so far.

We see there's a role for initiation factor 1.

And in this cartoon, if we imagine the E site,

the P site, and the A site, what we see

is that IF1 is binding to the site of the ribosome.

And one way we can think about this

is that the initiator tRNA has to get to the P site.

And so that region is blocked to facilitate the initiator

tRNA getting to the P site.

OK, we see that initiator tRNA binding to the P site.

And this happens via formation of a ternary complex

with IF2 and GTP.

So initiation factor 2 hydrolyzes GTP.

There's an event that results in joining of the two subunits.

And there has to be dissociation of these initiation

factors for the ribosome to be ready to accept

its first aminoacyl-tRNA in the A site.

OK, so the outcome of this process

here is that we have an assembled 70S

ribosome with the initiator tRNA in the P site.

The A site is empty, so it can accommodate

an incoming aminoacyl-tRNA.

And the E site or exit site is also empty.

So that's the main take home for initiation.

And that's the extent to which we're

going to discuss it within this class.

So in order to get to the elongation cycle,

we need to get the aminoacyl-tRNA into the A site.

And that's going to require the help of EF-Tu, so elongation

factor Tu.

Before we discuss how elongation factor

Tu is going to help deliver that aminoacyl-tRNA,

we need to talk about how we get the aminoacyl-tRNA

in the first place.

So what is the tRNA structure, just as a review

to get everyone up to speed.

How are amino acid monomers attached to the tRNA?

And how is the correct amino acid attached?

So this is an aspect of fidelity,

which came up as a concept last week in lecture.

And so we'll look at the mechanism

of aminoacyl-tRNA synthetases to see

how is the correct amino acid attached,

and then what happens if the wrong amino acid is selected.

Are there mechanisms to correct that?

And if it's not corrected, what are the consequences here?

So moving forward with that, we're

going to focus on the tRNAs and addressing those questions.

So just as a review, so we can think

about tRNA secondary structure, which is often

described as cloverleaf.

So we have a five prime end.

The tRNA has several arms.

OK.

So we have a D arm.

This arm here has the anticodon that pairs

with the codon of the mRNA.

We have a variable arm, this arm here.

And we have this three prime end here,

where the amino acids get attached.

So this, in terms of base numbering,

we have C74, C75, A76 here.

OH.

This is often called the CCA acceptor stem.

And the amino acids are attached here.

I'm going to abbreviate amino acid

as AA via an ester linkage.

And these ester linkages are important for the chemistry

that happens in the ribosome.

OK.

So we can imagine just if we have abbreviating the tRNA

structure like this and if we think about the sugar of A76--

bless you.

OK.

We have one prime, two prime, three prime here.

This type of connectivity here.

And this is abbreviated throughout as amino acid tRNA,

aa in general terms, or the three-letter abbreviations,

like what we saw for f-met tRNA f-met with the initiator tRNA

here.

So here's a schematic of a tRNA secondary structure with a bit

more detail than what I show you on the board.

And something we need to keep in mind

is even though we often draw the tRNA in this cloverleaf type

depiction, it has tertiary structure.

And so it's very important to think about this structure

as we think about how the tRNAs enter the various sites

of the ribosome.

OK.

So this structure is L-shaped.

And I like this depiction here because regions

of the secondary structure are color

coded with the corresponding regions

of this tertiary structure here.

OK, so we see the shell shape of an L,

rather upside down here, where we have the CCA acceptor stem

over here and the anticodon arm and anticodon region down here.

So what is a consequence of this structure?

The tRNA is quite narrow.

So we're thinking about 20 to 25 Angstroms in width.

And if we think about this in the context of the ribosome

and the peptidyl transferase center,

3 tRNAs need to fit into that catalytic center

during the elongation cycle.

So it makes sense that they're relatively narrow.

This allows three to fit there.

So as we think about the translation process

and also think about some of the translation factors,

we want to keep this type of structure in mind here.

Here's just another view of that,

with some additional descriptions

of the overall structure.

And this includes the numbering of the tRNA bases

within that structure here.

Just a point to make, this won't be a major focal point

in the course, but do keep in mind

that tRNA contains many post-transcriptionally modified

bases, so you'll see an example of that in problem set one.

Up to 25% of the bases can be modified.

Typically, we see about 5% to 20% of them modified here.

OK, you're not responsible for these structures,

these modified structures, in the context of this class.

So the key question for today is how

are amino acids attached to the tRNA, as shown here?

And in order for that to happen, there's

a family of enzymes called aminoacyl-tRNA synthetases,

or abbreviated aaRS.

OK, so this name tells you right away, synthetase,

that these enzymes use ATP.

And these enzymes catalyze the attachment

of amino acids to the three prime OH,

or sometimes two prime OH, of the tRNA here for that.

And so we're going to consider this overall reaction.

And then we're going to think about the reaction

mechanism and experiments that were done to give support

to the mechanism that we see.

So all aminoacyl-tRNA synthetases

require ATP and hydrolyze ATP to AMP and PPI.

And so they catalyze this overall reaction

where we have an amino acid monomer.

We have the tRNA that encodes this--

that is for this amino acid.

ATP to give us the aminoacyl-tRNA AMP and PPI.

So if the ATP is being hydrolyzed to AMP and PPI,

what phosphate is being attacked?

So we saw on Friday there's the alpha, beta,

and gamma phosphates of ATP.

OK, pardon?

AUDIENCE: Beta.

ELIZABETH NOLAN: Beta.

Any takers?

AUDIENCE: Alpha?

ELIZABETH NOLAN: Any takers?

Gamma?

Yeah, so it's alpha.

If you're getting AMP, it's attack at alpha.

If you're getting ADP, it's attack at gamma here.

OK, so P alpha is next door to the ribose of the nuc--

there.

Yeah.

OK.

So if we consider this overall reaction, how does it work?

Just before that, one other observation

I just want to point out, if we're

thinking about these enzymes and asking

what is it that they recognize of the tRNA,

so we have the anticodon.

And that goes in hand-in-hand with the identity

of the amino acid.