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  • ELIZABETH NOLAN: So last time, we

  • were talking about these aminoacyl tRNA synthetases that

  • are responsible for attaching amino acid

  • monomers to the three prime end of tRNAs.

  • And we were looking at the isoleucyl aminoacyl tRNA

  • synthetase as an example, looking at experiments that

  • were done to study mechanisms.

  • So recall, we left off having discussed a two-step model,

  • where there's an intermediate, an amino adenylate formed.

  • And then, in the second step, there's

  • transfer of that amino acid to the tRNA by the aaRS.

  • And so we looked at some data from steady-state kinetic

  • experiments.

  • Recall that a C14 radiolabel was used to watch transfer,

  • and then we closed discussing an ATP-PPi exchange assay which

  • gave evidence for formation of that amino adenylate

  • intermediate.

  • Right?

  • And then, lastly, we talked about use of a stopped-flow

  • to do experiments that allow you to look at early points

  • within a reaction.

  • And so what we're going to do is to close these discussions

  • of experiments and this aaRS mechanism

  • is just look at one more experiment that

  • was done to further probe the rate-determining step

  • of this reaction using the stopped-flow.

  • OK?

  • And so this experiment pertains more to reaction kinetics,

  • and the question is, let's monitor

  • transfer of the amino acid to the tRNA

  • by another method here.

  • These experiments were set up in two different ways

  • depending on what components were mixed.

  • And if you just rewind to Monday and recall the ATP-PPi exchange

  • assay and the steps in that assay, in that we showed

  • that the amino adenylate intermediate remained

  • bound to the enzyme there.

  • Recall then only PPi was released in that assay.

  • And so in these experiments, the fact

  • that the amino adenylate can remain bound

  • was taken advantage of.

  • And the researchers were actually

  • able to have a preformed complex there, so basically

  • starting after step two.

  • So in experiment one, how I'm going to show

  • these is by drawing the two syringes

  • and listing the components of each syringe.

  • And this is a good way for setting up problems

  • within the problem sets, thinking

  • about stopped-flow experiments.

  • So the question is what are we going to mix?

  • So we have syringe one and syringe two,

  • and recall that these go to some mixer.

  • So the two solutions can be rapidly mixed,

  • and that's where the chemistry is going to happen.

  • So in experiment one, in syringe one,

  • what we have is the purified complex.

  • OK?

  • So we have C-14 labeled isoleucine-AMP

  • bound to the aminoacyl tRNA synthetase

  • of a purified complex, here.

  • And then in this other syringe two, what we have is the tRNA.

  • OK?

  • So imagine these are rapidly mixed.

  • There'll be transfer of the radiolabeled isoleucine

  • to the tRNA, and so formation of that aminoacyl tRNA

  • can be monitored.

  • OK?

  • In the second experiment, we have just theme in variation,

  • and if you're interested in more details,

  • the reference is provided in the slides.

  • So again, in syringe two, we have the tRNA,

  • and in syringe one, what will be combined

  • are the components here.

  • OK?

  • So then, the question is, in each case, what do we see?

  • And those data are presented here from the paper,

  • and there's some additional details

  • about the experimental setup.

  • So effectively, what we're looking at on the y-axis

  • is the amount of tRNA that's been modified.

  • So tRNA acylation measured by transfer

  • of the radiolabel versus time.

  • And in the black circles, we have the data

  • from experiment one, shown here, and in the open circles,

  • we have the data from experiment two.

  • So what is the conclusion from these data?

  • And this value here is not similar to something we've

  • seen before in this system.

  • Both experimental setups are giving the same result. Right?

  • Effectively, these data are superimposable,

  • and they can be fit the same.

  • So what does that tell us about the rate-determining step?

  • AUDIENCE: [INAUDIBLE] versus forming the intermediate.

  • ELIZABETH NOLAN: Yeah.

  • Right.

  • Aminoacylation of tRNA is the rate-determining step.

  • So some of you suggested that in class on Monday.

  • Right?

  • So that's the case here.

  • OK?

  • So formation of the intermediate is much more rapid

  • than acylation of the tRNA here.

  • So we've examined now the mechanism

  • in terms of getting the amino acid onto the tRNA.

  • What do we need to think about next here?

  • So what we need to think about is fidelity.

  • OK, and we've looked at the overall rate of error

  • in protein biosynthesis, how often errors occur

  • on the order of 10 to the 3.

  • So how is the correct amino acid loaded onto the correct tRNA?

  • Each tRNA has an anticodon that is a cognate pair with a codon.

  • And so different tRNAs need to have

  • different amino acids attached.

  • OK, and what does that mean?

  • That means, in general, there's a dedicated aminoacyl tRNA

  • synthetase for each amino acid, in general here.

  • So how are amino acids with similar side chains

  • differentiated by these enzymes?

  • And is it possible for an incorrect amino acid

  • to get loaded onto a tRNA?

  • And if that happens, what are the consequences?

  • So we're going to examine fidelity some here.

  • And as background, an observation made,

  • say from studies like that ATP-PPi exchange assay,

  • is that some aminoacyl tRNA synthetases can activate

  • multiple amino acids, so not only the one

  • they're supposed to activate but also others.

  • So what does that mean?

  • That means that the enzyme can bind

  • and activate effectively the wrong amino acid,

  • and if we think about fidelity, we

  • can think about this as being a problem here.

  • So what happens?

  • What happens is that these enzymes have an editing

  • function, and they're able to sense if a wrong amino acid is

  • activated.

  • And then they have a way to deal with it,

  • and this is by hydrolysis.

  • OK?

  • And so let's consider an example, for instance, just

  • similar side chains.

  • So if we just consider, for instance, valine, isoleucine,

  • and threonine, these will be the players for our discussion.

  • OK?

  • They're different, but they're not too different.

  • Right?

  • Oops, sorry about this.

  • We're missing a methyl.

  • Valine, an isoleucine, we have a difference of a methyl group.

  • Threonine, we have this OH group.

  • Right?

  • And we can just ask the question,

  • for instance, how is valine differentiated from isoleucine

  • or threonine here?

  • And so as an example, what's found

  • is, if we consider our friend that we studied

  • for the mechanism here, what we find

  • is that this binds and activates isoleucine, as we saw,

  • but it will also bind and activate valine here.

  • And effectively, if this happens,

  • we have a mismatch, because the end result

  • will be isoleucine-RS with valine AMP bound here.

  • OK?

  • And what's found is that the catalytic efficiency or Kcat

  • over Km, in this case, is about 150-fold

  • less than the native substrate.

  • So that doesn't account for the 10 to the 3 error rate here.

  • So we need more specificity.

  • So what's going on?

  • So we're going to consider this editing function and a model

  • that's often used to describe how these aaRS do

  • editing is one of two sieves.

  • These enzymes don't actually have a sieve.

  • It's just a conceptual way to think about it.

  • So this double-sieve editing model

  • involves a first sieve which is considered to be a course one.

  • So imagine if you have like a change sorter.

  • It will let the quarters through as well as

  • the and dimes and the pennies.

  • There's some sort of discrimination

  • of amino acids based on size, and then

  • depending what gets through this first sieve or gate,

  • there's a second sieve which is considered to be a fine one.

  • And this one can differentiate perhaps on the basis of size

  • or maybe on hydrophilicity or hydrophobic of the side chain.

  • So effectively, if an incorrect amino acid passes through this

  • first sieve-- so in other words, if it binds to the enzyme

  • and becomes activated--

  • hydrolytic editing will occur.

  • OK?

  • So think about hydrolysis in terms of having

  • breakdown of these species.

  • So if the incorrect amino acid passes through

  • and is adenylated, there'll be hydrolysis.

  • So let's consider some examples so the first example here we

  • can consider this guy and isoleucine and valine.

  • So as I mentioned, this aaRS will activate both.

  • So in this case, the first sieve can't differentiate isoleucine

  • from valine.

  • They have similar sizes according to this aaRS.

  • But then what happens here in the second sieve,

  • isoleucine is too big, and so there's no hydrolysis,

  • and it moves on to form the desired charged tRNA.

  • In contrast, valine's a bit smaller.

  • It passes through the sieve, and it ends up being hydrolyzed.

  • So these aaRS also have an editing domain,

  • and this editing domain, as we'll

  • see in a few slides in a structure,

  • is responsible for this hydrolysis, so stated here.

  • Right?

  • Different sites, so there's an aminoacylation site

  • and an editing site here.

  • So valine can reach the editing site, but isoleucine cannot.

  • So how do you predict?

  • Just to keep in mind, every enzyme

  • is different in terms of the model

  • for discrimination and also when editing occurs.

  • So you really need to look at the data

  • when the data is presented to you to sort out how this works.

  • Let's just look at another example

  • with a cartoon depiction.

  • So this is for the valine RS, and we're

  • going to consider the three amino acids here--

  • valine, threonine, and isoleucine.

  • So in green, we have the first sieve,

  • and this is based on size.

  • So what do we see in this cartoon?

  • So threonine and valine make it through,

  • but isoleucine does not.

  • It's rejected right away, so it's never activated.

  • So if threonine and valine pass through, what happens?

  • We see each one is activated as the amino adenylate, and then

  • what?

  • Well, valine, we want to transfer the valine

  • to the tRNA, so it can move on and help

  • with protein synthesis.

  • If threonine's activated, and here we

  • see that threonine is transferred to the tRNA

  • as well, this is hydrolyzed by the editing site, in this case.

  • So the threonine is removed from the tRNA

  • with the anticodon for valine.

  • Right, so think about the ester bonds

  • that we saw last time in terms of the three prime end

  • of the tRNA being modified and the chemistry that

  • will happen there to result in hydrolysis of and release

  • of the amino acid here.

  • So what that cartoon hints to is that the hydrolysis can

  • occur at different steps.

  • So we can have hydrolysis that is pre-transfer,

  • which means the editing occurs before the tRNA is modified.

  • Or we can have post-transfer editing

  • which is what we saw in the prior slide, where

  • the editing and hydrolysis occurs after the amino acid

  • monomer is transferred to the tRNA.

  • OK?

  • And this schematic here depicts that, so what do we have?

  • We have the aaRS responsible for modifying tRNA for isoleucine,

  • and we combine that with valine, the wrong amino acid, and ATP.

  • What happens?

  • So E is for enzyme.

  • We have formulation of the amino adenylate intermediate.

  • Here's the tRNA with the anticodon for isoleucine.

  • What happens?

  • So we have this complex form in this depiction.

  • Pre-transfer editing would occur at this stage,

  • before the valine is transferred to the tRNA, and so

  • what do we see?

  • We see breakdown and these species.

  • If the valine is transferred to the tRNA,

  • we don't want this, because that would

  • result in this reading of the genetic code.

  • Post-transfer editing, this species here is hydrolyzed.

  • So whether pre or post-transfer editing occurs

  • is going to depend on the aminoacyl tRNA synthetase,

  • and some can use both mechanisms.

  • That's what we're seeing here.

  • OK?

  • Some only use one, for instance, the valine RS

  • only uses a post-transfer editing mechanism.

  • So when presented with the data, look at the data

  • and see what species is being hydrolyzed.

  • And if both are, how did the steady-state kinetics,

  • for instance, compare?

  • Just to take a look in the context of a structure of one

  • of these aaRS.

  • So the sites where aminoacylation

  • occur and editing occur are separated

  • by about 30 Angstroms, and that's

  • shown here, where we have the aminoacylation site,

  • and here we have the editing site.

  • That's responsible for pre and/or post-transfer editing.

  • So in thinking about this and thinking

  • about how one could leverage this 30 Angstrom

  • separation and these two distinct sites

  • in terms of experiments, what does that allow one to do?

  • So imagine if you want to ask, what

  • are the consequences of having aaRS that have faulty editing

  • function?

  • And effectively, mischarged tRNAs

  • or put the wrong amino acid on a tRNA.

  • What does that mean for a cell?

  • There's an opportunity to do that here.

  • So you could imagine mutating residues

  • that are critical for editing function in the editing site.

  • Such that you have an aaRS variant that can activate amino

  • acids and transfer them to the tRNA but cannot edit when

  • a mistake happens.

  • Right?

  • So you can imagine a site-directed mutagenesis,

  • purifying the enzyme and doing some in vitro characterization

  • to see how it behaves.

  • And then you could also imagine translating this

  • into a cellular context and asking say in cell culture what

  • happens here?

  • So basically, what are the consequences of faulty editing?

  • And these types of studies have been done.

  • We're not going to look at them in detail.

  • But just as an overview and some concepts that will come up

  • within our folding section, what's been shown

  • is that a single point mutation in an editing domain of one

  • of these aminoacyl tRNA synthetases

  • may have deleterious consequences.

  • And we can imagine that these consequences could result

  • from proteins or enzymes that gain a new function

  • or don't do their correct function.

  • Right?

  • So just imagine that some mischarged tRNAs, where

  • mischarged means the wrong amino acid is attached,

  • are around because of some mutant aaRS.

  • And these tRNA that are mischarged

  • can be delivered to the ribosome, which

  • means that point mutations form within synthesized polypeptide

  • chains.

  • So there's some mixture where some of these proteins

  • are native, and others are mutant,

  • and what might happen here in terms of consequences?

  • So native protein will go on and do its job.

  • Imagine there's some mutant protein here

  • that's altered in some way, and these are just some examples

  • of possible outcomes.

  • So maybe there's a breakdown of some essential cellular

  • process.

  • Here, we have triggering of autoimmune-like responses,

  • things that are not good.

  • What if these mutant proteins misfold?

  • So they can't form their correct fold,

  • and fold is important for function.

  • Maybe there's aggregation.

  • Maybe there's stress on the proteasome, ER response,

  • unfolded protein response, cell death.

  • So fidelity's important.

  • And just some things to think about as we close this section.

  • We can consider error rates of various biological

  • polymerizations, whether that be DNA replication, transcription,

  • or translation, and they vary quite a bit here from this.

  • And what the take-home can be by comparing these error

  • rates is infrequent mistakes in decoding the mRNA

  • are accepted as a source of infidelity.

  • So they do occur, and they occur more frequently

  • than, say, an error in replicating the DNA,

  • and that makes sense.

  • Right?

  • If an error occurs in DNA replication,

  • there's a huge problem likely compared

  • to an error in translation.

  • So some questions just to think about,

  • answers aren't going to come up within the context

  • of this course.

  • But higher accuracy is important, but actually

  • how much accuracy is enough?

  • And there is a cost in terms of cellular energy for accuracy,

  • and is it that the cell tunes its accuracy to some point that

  • could be considered optimal, and are

  • there benefits to translational infidelity?

  • Right?

  • So the prior slide showed negative consequences,

  • but are there benefits?

  • So that discussion, we'll close considering

  • how the amino acids get attached to tRNAs,

  • and so where we're moving to now is the elongation cycle.

  • AUDIENCE: So is there a specific part of the cytoplasm

  • where the tRNAs and the amino acids

  • come together, or does this happen everywhere?

  • ELIZABETH NOLAN: So I actually don't know,

  • but I think of them as being everywhere

  • in terms of the tRNAs.

  • Because as we'll see in a few slides,

  • EF-Tu, which is required for delivering

  • the tRNAs to the ribosome, is highly abundant.

  • At least, that's my thinking for prokaryotes.

  • Do you have anything to say?

  • The question was effectively are there

  • certain regions of the cell where tRNAs get modified more

  • than other regions?

  • JOANNE STUBBE: I don't know.

  • In mammalian cells, they have weirdo complexes

  • with tRNA synthases that they've been around forever.

  • and I still think we don't really understand

  • what the function is.

  • AUDIENCE: [INAUDIBLE]

  • JOANNE STUBBE: Can you speak a little bit louder?

  • ELIZABETH NOLAN: The question is, do we

  • have information about say the distribution of tRNAs

  • as being amino acid modified versus unmodified?

  • AUDIENCE: I think maybe we could [INAUDIBLE] I don't know.

  • ELIZABETH NOLAN: There's always a way, probably.

  • Right?

  • But I don't know what that distribution is either

  • in terms of the percentage of tRNAs that are aminoacylated

  • at any one given time.

  • Yeah, just don't know.

  • I think one key thing to think about

  • as we come to the next part is that these tRNAs are

  • bound by EF-Tu.

  • So to think of them as in complex with a translation

  • factor as opposed to tRNAs floating around

  • in the cytoplasm, so I think that that's

  • a key point of focus.

  • So moving into elongation, what do we need to think about here?

  • So we need to think about delivery

  • of the amino acid tRNAs.

  • How does the ribosome ensure that the correct aminoacyl tRNA

  • is delivered?

  • So we have the correct amino acid onto the tRNA,

  • but we also have to get the correct amino acid

  • to the ribosome.

  • How is peptide bond formation catalyzed?

  • What is the method by which polypeptides

  • leave the ribosome, and how is translation terminated here?

  • So effectively, these are all questions

  • we need to address in terms of thinking about how

  • the ribosome translates the genetic code

  • and synthesizes the polypeptide.

  • So within the notes posted on Stellar,

  • there's a number of pages of definitions, so terminology

  • that comes up within these discussions of the ribosome

  • to refer to.

  • And in terms of our translation overview slide,

  • where we are now is here, in elongation.

  • So we have the mRNA our 70S, and we're

  • going to focus for the rest of today on thinking about EF-Tu,

  • this elongation factor that's responsible for delivering

  • the amino acid tRNAs to the ribosome here.

  • So as an overview in terms of a cartoon, where are we going?

  • Here, we have our ribosome, and in this depiction,

  • it has been translating.

  • So we have a nascent polypeptide emerging through the exit

  • tunnel of the 50S.

  • So we see this peptidyl tRNA in the P-site,

  • and we have this deacylated tRNA in the E-site.

  • So what happens?

  • That A-site is empty, and for another round of elongation

  • to occur, the aminoacyl tRNA needs to be delivered.

  • And as we'll see today and in recitation this week,

  • EF-Tu is responsible for that.

  • So there's a ternary complex that forms between EF-Tu-GTP.

  • So EF-Tu is a GTPase and the aminoacyl tRNA.

  • And this ternary complex delivers the aminoacyl tRNA

  • to the A-site.

  • OK?

  • This allows for peptide bond formation

  • to occur in the catalytic center.

  • And then there's a process called translocation,

  • in which the elongation factor-G in complex with GTP

  • comes in and helps to reset the ribosome such

  • that another aminoacyl tRNA can come in.

  • So where we're going to focus for the rest of today

  • is on this process here, thinking about EF-Tu

  • and how that delivers amino acid attached

  • to tRNAs to the A-site.

  • OK, so just in our cartoon, where we left off,

  • with initiation process, so we have

  • that initiator tRNA in the P-site,

  • and the A-site is empty.

  • OK?

  • And one other thing I'll just show here,

  • I mentioned when describing ribosome structure

  • that some ribosomal proteins have additional jobs.

  • So it's not just that these proteins

  • help with the overall structural integrity of the ribosome.

  • And there's two ribosomal proteins, L7 and L12,

  • and these are involved in recruitment

  • of that ternary complex between EF-Tu, the GTP,

  • and the aminoacyl tRNA.

  • So now, we need to get the aminoacyl tRNA to the A-site,

  • and this requires EF-Tu.

  • And when we think about this, we always

  • need to think about this ternary complex which

  • is EF-Tu bound to the aminoacyl tRNA bound to GTP.

  • So a little bit about EF-Tu.

  • So in E. coli, EF-Tu is the most abundant protein.

  • So there's tons of EF-Tu.

  • OK, approximately here, we have 100,000 copies per cell.

  • So it's about 5% of total cellular protein.

  • And so, as I just said in response

  • to a question about these tRNAs in the cells,

  • we can think about this entire tRNA

  • pool, or aminoacylated tRNA pool,

  • as being sequestered by EF-Tu.

  • So EF-Tu binds the aminoacyl tRNA,

  • and it binds GTP to form the ternary complex.

  • And this allows EF-Tu to deliver these amino acids attached

  • to the tRNAs to the A-site, and it's a GTPase.

  • And we need to think a lot about how this activity relates

  • to its function and fidelity.

  • So here is a depiction of the structure of a ternary complex.

  • So what we see is that we have a tRNA here,

  • and here we have EF-Tu bound to the tRNA.

  • So here is the anticodon loop, and if we consider

  • this structure of the ternary complex bound to mRNA,

  • what do we see?

  • So we have an mRNA in green.

  • OK, here's the tRNA, and the anticodon end,

  • and here's EF-Tu.

  • And as I said, EF-Tu is a GTPase.

  • Where is the GTPase center?

  • That's up here.

  • So this GTPase center of EF-Tu is quite far from the tRNA

  • anticodon, down here.

  • This distance is about 70 Angstroms.

  • And so this is something quite incredible

  • to think about, because as we'll see,

  • when there's codon recognition--

  • meaning this codon-anticodon interaction,

  • that's a cognate pair--

  • GTP hydrolysis is stimulated.

  • So how is that communicated over 70 Angstroms?

  • If there's a recognition of that here

  • between the mRNA and the tRNA anticodon,

  • and GTP hydrolysis happens up here,

  • how is that signaled over 70 Angstroms?

  • Right?

  • So clearly, there's going to be some conformational changes

  • that occur that allow this GTPase activity to turn on.

  • Just another view, so here, again, we

  • have the structure of the ternary complex bound

  • to the mRNA, and here, we can look at the ternary complex

  • bound to a 70S ribosome.

  • So we have the ribosome in this orangey-gold color,

  • the 50S the 30S.

  • Here, we have the PTC and decoding site.

  • The tRNA is in green, and EF-Tu is in this darker orange here,

  • to place that in the perspective of the 70S ribosome here.

  • So conformational change is required

  • to signal code on recognition to the GTPase center,

  • and this is something that will be

  • spoken about in quite some detail this week in recitation.

  • One other point of review before moving forward with delivery

  • of the amino acid tRNA.

  • We need to think about codon-anticodon interactions

  • here for decoding.

  • So we have cognate versus near-cognate

  • versus non-cognate, and this is for the codon-anticodon

  • interaction.

  • OK, and so if we imagine we have some mRNA,

  • and you need to think about the five prime and three prime ends

  • with this.

  • And then we have some tRNA, three prime, five prime,

  • we need to ask how do these match?

  • So for instance here, if we have AAG,

  • and we have positions one, two, three,

  • from left to right of the mRNA, right here

  • we have a cognate match.

  • OK?

  • So we have the AU match in positions one and two,

  • and then wobble's allowed in position three, this GU here.

  • So no, no interaction.

  • OK, just as another example here,

  • imagine we have GAG, here.

  • What we see is that there's only one

  • match, meaning Watson-Crick base pairing, in position two.

  • OK.

  • Here, this GU, that's not a match

  • based on Watson-Crick base pairing, and as a result,

  • the ribosome is going to want to reject this tRNA,

  • if this is what's happening in the A-site here.

  • And then, we can just imagine some situation,

  • where we have a tRNA and an mRNA where there's just no match.

  • OK?

  • No Watson-Crick base pairing here.

  • So what we need to ask is, as EF-Tu is delivering

  • these aminoacyl tRNAs, what happens

  • if it's a cognate match versus a near-cognate

  • versus a non-cognate?

  • How does the ribosome deal with the wrong tRNA entering

  • the A-site?

  • Right?

  • So again, this is something important for fidelity,

  • and these both need to be rejected.

  • So why are we reviewing this?

  • We're reviewing this, because it's

  • important in terms of what happens

  • during initial binding of aminoacyl tRNAs

  • to the ribosome.

  • So we're going to go over some of this in words

  • and then look at a cartoon that explains this process.

  • And what we're focused on is delivery of the aminoacyl tRNA

  • to the A-site.

  • So what happens first?

  • OK.

  • First, there needs to be an initial binding

  • event, where the ternary complex binds to the ribosome.

  • So initial binding, it binds to the 70S,

  • and these ribosomal proteins are involved in the recruitment

  • of the ternary complex.

  • This initial binding event of the ternary complex

  • to the ribosome is independent of the mRNA.

  • What happens next is that there's codon recognition.

  • So we need to think about that tRNA entering the A-site,

  • and there's some sort of sampling

  • that occurs in the decoding center, so

  • sampling of codon-anticodon pairs in the A-site,

  • and so what happens?

  • What happens if there's a cognate event

  • or a non-cognate event?

  • So if a cognate anticodon recognition event occurs,

  • there's a series of steps that then happen.

  • So with a cognate codon-anticodon interaction,

  • there will be a conformational change in EF-Tu,

  • and this activates the GTPase center which

  • allows for GTP hydrolysis.

  • OK, and effectively this conformational change

  • stabilizes the codon-anticodon interaction here,

  • and that stabilization accelerates the GTP hydrolysis

  • step.

  • So this is all building towards a kinetic scheme.

  • In terms of enhancements, what's found is that the rate of GTP

  • hydrolysis by EF-Tu increases by about 5 times 10

  • to the 4th with cognate anticodon

  • recognition in the A-site.

  • So we have GTP hydrolysis, and then there's

  • another conformational change.

  • So we have EF-Tu in its GDP-bound form,

  • and effectively, EF-Tu will dissociate

  • from the aminoacyl tRNA, and the aminoacyl tRNA will fully

  • enter the A-site.

  • OK so this process is called accommodation,

  • and once that happens, peptide bond formation can occur.

  • So this is the good scenario.

  • The polypeptide can keep being made.

  • What if it's not a cognate?

  • So what if a near-cognate tRNA is delivered to that A-site

  • during this initial binding event which

  • is independent of the mRNA?

  • That's why this can occur.

  • If it's a near-cognate anticodon, what we observe--

  • and this is all from experiments you'll

  • be learning about this week--

  • the ternary complex rapidly dissociates from the ribosome.

  • And what's found from kinetic measurements

  • is that the dissociation of the ternary complex,

  • when it's a near-cognate situation,

  • is about 350-fold faster than cognate.

  • So let's look at this stepwise within a cartoon format.

  • You'll see another depiction of this scheme

  • in the recitation notes and in problem set two.

  • So here, we have multiple steps in this overall process.

  • All of these steps have some rate

  • that's been measured by multiple types of methods,

  • and Joanne will be presenting this week

  • on a lot of pre-steady-state kinetic analysis that were done

  • to measure these rates here.

  • And basically, the key point to keep in mind, and that I'd

  • like to stress from what was just said on the prior slide,

  • is that what you'll see throughout this

  • is that conformational changes are coupled

  • to these rapid chemical steps.

  • And the chemical steps are irreversible,

  • this GTP hydrolysis.

  • So what do we see?

  • We begin with initial selection.

  • Here, we have our ribosome, and there's a polypeptide

  • being synthesized.

  • Here's the ternary complex--

  • EF-Tu, GTP, and the aminoacyl tRNA.

  • So there's an initial binding step

  • that's governed by k1 in the forward direction and k minus 1

  • in the back direction, and said before, this

  • is independent of the mRNA.

  • So what happens?

  • The ternary complex binds the ribosome,

  • there's sampling in the A-site of the anticodon,

  • and then there is a step described as codon recognition

  • with k2 and k minus 2.

  • OK?

  • In this scheme, if an arrow is colored,

  • red arrow indicates the rate is greater

  • for near-cognate than cognate.

  • OK?

  • Which means in the event here of a cognate pair,

  • this is going to push forward in the forward direction.

  • If it's near-cognate, this back step

  • has a greater rate of about 350-fold.

  • OK?

  • So we're going to end up back here.

  • With cognate recognition, next, we

  • have GTPase activation, again, forward and reverse.

  • Green indicates the rate is greater for a cognate match

  • than near-cognate.

  • So if it's the correct anticodon,

  • it's going to plow through to here.

  • We have GTPase activation.

  • And then what happens down here?

  • We have a GTP hydrolysis step.

  • We have a conformational change in EF-Tu, and then what?

  • We can have accommodation such that the tRNA was installed

  • fully into the A-site and then rapid

  • peptide bond formation or peptidyl transfer.

  • The ribosome has one last chance to correct a mistake.

  • So you can imagine that after GTP hydrolysis,

  • after the conformational change in EF-Tu and its dissociation,

  • there's a last chance at rejection here.

  • Realize that step is occurring at the expense of GTP here.

  • So in thinking about how to deconvolute this model

  • or how to design experiments to test this model,

  • there's a lot that needs to be done.

  • Right?

  • A lot of rates that need to be measured,

  • a lot of different species along the way with the ribosome.

  • Right?

  • So how do you get a read out of each of these steps?

  • That's what we'll be focused on in recitation this week

  • and next here.

  • So here are some more details on this initial binding process

  • with some information related to the k1s and k minus 1s here.

  • That's provided to help navigate the reading this week

  • for recitation here.

  • So what happens in the GTPase center of EF-Tu?

  • What are some of these conformational changes?

  • And effectively, there are conformational changes

  • in the decoding center that are critical on one hand.

  • So that's not at the GTPase center,

  • but first asking what's happening when the mRNA

  • and tRNA codon interact?

  • And then what's happening in the GTPase center here?

  • So just to note, not shown in the slide in terms

  • of the decoding center.

  • OK, what we need to be focusing on are changes in the 16S RNA,

  • and effectively, I'll just point out three of the positions.

  • So we have A1492, A1493, and G530 of the 16S, here.

  • And what we find is that these bases effectively

  • change conformation with a cognate match.

  • And they effectively flip and interact

  • with that cognate anticodon to help

  • stabilize the codon-anticodon interaction.

  • So this stabilizes the codon-anticodon interaction,

  • and that stabilization accelerates the forward steps.

  • So that results in this acceleration of GTP hydrolysis.

  • So then the question is, what's happening

  • in the GTPase center of EF-Tu?

  • Because there has to be a change in conformation at that GTPase

  • center 70 Angstroms away to allow for GTP

  • hydrolysis to occur, and somehow,

  • that all has to be signaled from here to there.

  • So what we're looking at here is an excerpt

  • of the structures looking at this GTPase center,

  • and so what do we see?

  • Effectively, two residues, so isoleucine-60 and valine-20

  • have been described as a hydrophobic gate in the GTPase

  • center.

  • OK, and the idea is that if this gate is closed,

  • it prevents a certain histidine residue, histidine-84,

  • from activating a water molecule which then allows for the GTP

  • to be hydrolyzed.

  • OK, but if there's a change in conformation,

  • and this gate opens, that chemistry can occur.

  • So what are we looking at here in these structures?

  • Effectively here, we have the two hydrophobic residues

  • of the gate, so valine-20, isoleucine-60,

  • and here's that histidine-84 I told you about,

  • and what is this, GTPCP?

  • So what we have there is a nonhydrolizable GTP analog.

  • These types of molecules are very

  • helpful in terms of getting structural information,

  • in terms of doing certain types of biochemical experiments.

  • OK?

  • So effectively, we can have an analog bound that cannot

  • hydrolyze.

  • What are we looking at here?

  • Here, we're looking at the, say, activated species,

  • and what do we see?

  • We see that this histidine has changed position.

  • So here, it's flipped that way, here this way and here,

  • what we see is a view with EF-Tu in the GTP-bound form.

  • So the idea is that overall conformational changes that

  • occur 70 Angstroms away, because of codon-anticodon recognition,

  • effectively signal conformational changes

  • in GTPase center that allow for GTP hydrolysis to occur

  • and things to move in the forward direction there.

  • So that's where we'll close for today.

  • On Friday, we'll continue moving forward in this elongation

  • cycle, and starting in recitation tomorrow,

  • you'll look at experiments that allowed for this kinetic model

  • to be analyzed and presented.

  • You really need to come to recitation this week

  • and read the paper.

  • JOANNE STUBBE: And you need to read the paper more than once.

  • It's a complicated paper.

  • ELIZABETH NOLAN: That's on [INAUDIBLE]..

  • It's a complicated paper which is why we have

  • two weeks of recitation for it.

  • There's a lot of methods, and I'll also point out

  • that problem set three has very similar types of experiments,

  • but it's looking at EFG instead of EF-Tu.

  • So spending the time on this paper in the upcoming weeks

  • is really important.

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B1 中級

4.タンパク質合成 3 (4. Protein Synthesis 3)

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    林宜悉 に公開 2021 年 01 月 14 日
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