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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.