How Protein Factors Facilitate Protein Synthesis by the Ribosome
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00:00
NobeliumChemistry
00:29
NobeliumProteaseMultiprotein complexAmino acidHeck-ReaktionTransfer RNALibrary (computing)Lecture/Conference
01:21
NobeliumOrlistatGlucoseInduktorBinding energyConformational changeHydrolysatLecture/ConferenceMeeting/Interview
02:35
MoleculeDNS-SyntheseRNAProteinRibosomeDNS-SyntheseRNALecture/ConferenceMeeting/Interview
03:12
NobeliumMoleculeDNS-SyntheseRNAAmino acidTransfer RNAPipetteProteinNahtoderfahrungActive siteRibosomeTiermodellRNA
04:15
Peptidyl transferaseNahtoderfahrungPeptideProtein subunitSurface scienceIronSetzen <Verfahrenstechnik>ProteinPeptideComplication (medicine)RNADNS-Synthese
05:07
Protein biosynthesisPeptideRiver sourceChemical reactionSubstrat <Chemie>Transition metalAmino acidSetzen <Verfahrenstechnik>Protein biosynthesisRiver sourcePotenz <Homöopathie>RibosomeProteinChemical reactionBiochemistryComputer animationLecture/Conference
05:38
SlateChemical reactionPeptideSubstrat <Chemie>Transition metalAlpha particleSubstrat <Chemie>AmineAmino acidCarbon (fiber)Transfer RNAActive siteHydrocarboxylierungThermoformingGrowth mediumSpawn (biology)
06:03
RibozymeActive siteChemical reactionSubstrat <Chemie>RNAProteinLactitol
07:16
Active siteChemical reactionNobeliumProcess (computing)PeptideFunctional groupCarbon (fiber)Substrat <Chemie>Active siteAlpha particleAmineHydrocarboxylierungPotenz <Homöopathie>EntropyLactitolChemical reactionProteinPeriodateLecture/ConferenceMeeting/Interview
08:34
NobeliumProtein subunitActive siteLecture/ConferenceMeeting/Interview
09:02
Protein subunitActive siteSubstrat <Chemie>Substrat <Chemie>Man pageMeat analogueActive siteLecture/Conference
09:30
Active siteSubstrat <Chemie>Multiprotein complexNobeliumFunctional groupCocaineStorage tankOctane ratingCarbon (fiber)TaxisSubstrat <Chemie>Active siteHydrocarboxylierungConformational changeSkarnAlpha particleTeilentrahmte MilchLecture/Conference
10:19
TranslokationMultiprotein complexActive sitePeptideReaction mechanismProton-pump inhibitorFunctional groupWursthülleActive siteAmino acidMeat analogueSubstrat <Chemie>Binding energyProcess (computing)RibosomeGesundheitsstörungLecture/ConferenceMeeting/Interview
11:44
PeptideNobeliumReaction mechanismProton-pump inhibitorPeptideReaction mechanismProtonationMeeting/Interview
12:22
PeptideNobeliumReaction mechanismProton-pump inhibitorSet (abstract data type)Reaction mechanismAmino acidProtonationChemical reactionFunctional groupWalkingLecture/Conference
12:48
Multiprotein complexTransfer RNAWine tasting descriptorsMultiprotein complexLecture/Conference
13:13
Protein subunitPeptideActive siteSubstrat <Chemie>PhosphateMortality rateTetraederstrukturKohlenstoff-14Food additiveElectrical breakdownX-ray crystallographyProcess (computing)Carbon (fiber)Electrical breakdownMagnesiumProtonationMoleculeSide chainRibosomeActive siteWaterWalkingHydrocarboxylierungAmino acidFunctional groupSubstrat <Chemie>Teilentrahmte MilchGesundheitsstörung
15:28
Messenger RNARNATranslokationNobeliumMicroRNATransfer RNARustTranslation <Genetik>Cell (biology)AcidRiver sourceConformational isomerismPeptideTiermodellElektronenakzeptorMessenger RNAAzo couplingTranslokationChemical reactionTransfer RNATranslation <Genetik>TiermodellSubstrat <Chemie>Active siteCork taintMotion (physics)River sourceFalconryConformational changeThermoformingComputer animation
17:56
NobeliumTransfer RNAProtein domainTiermodellTranslokationHydrolysatRiver sourceCell (biology)Repeated sequence (DNA)AntibacterialElongation (astronomy)RibosomeConformational isomerismActive siteOptische AktivitätProtein subunitDeformation (mechanics)SunscreenElongation (astronomy)RibosomeSubstrat <Chemie>Active siteAntibacterialMessenger RNATransfer RNAChemical clockProtein subunitWalkingConformational changeOptische AktivitätBinding energyButcherFunctional groupWine tasting descriptorsTranslokationFiningsBlue cheeseTeaLecture/ConferenceMeeting/Interview
22:11
RibosomePeptideProteinTransfer RNAActive siteConformational isomerismTranslokationFunctional groupTranslation <Genetik>Electronic cigaretteResistenzReaction mechanismAntibacterialTransfer RNAPeptideActive siteLecture/ConferenceMeeting/Interview
23:41
NobeliumRibosomeAntibacterialMan pagePeptideAzo couplingTransfer RNAAmino acidMeeting/Interview
24:16
NobeliumRibosomeFunctional groupActive sitePeptideProtein subunitAntibacterialPeptideBinding energyAntibacterialActive siteGolgi apparatusZunderbeständigkeitMoleculeLecture/Conference
25:52
Ring strainMultiprotein complexAntibacterialPharmaceutical drugAntibiotic resistanceAntibacterialNobeliumPharmaceuticsX-ray crystallographyRNACrystallographyChemical compoundAntibacterialComputer animationLecture/ConferenceMeeting/Interview
26:51
NobeliumBarrel (unit)Stem cellEmission spectrumResistenzMacrolidePsychopharmakonActivity (UML)Phase (waves)Wine tasting descriptorsAntibacterialRecreational drug useChemical compoundSeparation processKlinisches ExperimentMeeting/InterviewComputer animation
27:57
CrystallographyCheminformaticsChemistryOxazolidinoneStem cellRing strainEmission spectrumPhase (waves)Klinisches ExperimentRing strainChemical compoundPrecursor (chemistry)ConcentrateSoilMinimale HemmkonzentrationLecture/ConferenceComputer animation
29:23
OxazolidinoneNobeliumStem cellRing strainAntibiotic resistanceMinimale HemmkonzentrationActivity (UML)AntibacterialBase (chemistry)Pharmaceutical drugFunctional groupSeleniteAnomalie <Medizin>Ring strainProcess (computing)WursthülleChemical compoundPharmaceuticsHope, ArkansasIceStop codonAdenineLecture/ConferenceComputer animation
31:21
NobeliumComputer animation
Transcript: English(auto-generated)
00:14
Well, it's a pleasure to be here again in this wonderful place and
00:21
Talk about our work on the on the ribosome, but I'll start a little earlier than that In 1968 Brian Hartley and enzymologists and
00:40
Cambridge LMB Suggested my first independent project. He came up to me in the hallway. He said tell me What are you going to do when you leave this? Your postdoc and start your own lab and I said well, I want to Solve the structures aminoacyl tRNA synthetase complex with tRNA
01:03
and amino acid He said there then my boy I suggest that you work on something you could actually do I Suggest you work on hexokinase So then I ran down the library to try and find out what the heck hexokinase was
01:21
There's Brian Hartley very good guy and It turned out that hexokinase had been One of the examples used by Brian Hartley to Sub for his induced fit theory
01:42
to explain why it is that Hexokinase doesn't hydrolyze ATP You have to have the glucose bind to cause a conformational change. So we saw the structures and and in fact the structure
02:00
Without Structure without ATP or a sort of hexo glucose Is open and then it closes down when glucose binds the other inspiration I had
02:21
In Cambridge was running into Francis Crick here And of course I'd run it I say of course I ran into Watson who is on the faculty at Harvard previously, so I knew these two chaps and I Got very interested in
02:42
Crick's central dogma, and so that's what I'm going to Talk about today Not the whole dogma that would take more than half an hour So this is central dogma that I'm sure you all know
03:03
DNA Is copied into DNA and Then the DNA is copied into RNA and then the ribosome Represented here. We'll have slightly higher resolution such shortly Attaches an amino acid from the A-site tRNA onto the P-site tRNA to elongate the polypeptide and
03:28
so We've been working on all these aspects, but I'm going to concentrate today on the ribosome and in 2000 of
03:42
I and my my colleagues were able Paul Nissen Menard bond in particular in Peter Moore. Oh we're able to get a structure and the talent model of the ribosome Was very exciting to see this very tightly packed RNA
04:02
None of us had any idea it was going to look like that With the active site being down at the bottom and with proteins embedded Throughout and if you split the ribosome in half You can see from the peptidyl transferase Center PT
04:24
Right down the middle. There's a tunnel and in that Tunnels about 100 angstroms long and in that tunnel is where the The polypeptide exits and then the whole ribosome the 70s
04:43
Consists of small subunit with the RNA threading around RNA is very complicated structure in the ribosome. It's not like DNA is not boring as as Jim might say And the proteins are embedded around on the surface
05:03
RNAs running around and the tRNAs are between the small subunit with the decoding It's going on and the large subunit where? With peptide bond formation is happening
05:21
So one of the questions is what's the power? What's the source of the ribosomes catalytic power and protein synthesis? The reaction which I'm sure you all know from your starting biochemistry classes Is the a site substrate has amino acid the alpha amino group attacks the carbonyl carbon of the peptidyl tRNA
05:47
Forms a tetrahedral intermediate and then goes to product. Well, what is the ribosome doing to make that happen? well we can stare into the
06:02
into the tunnel here and we can see Substrate analogs bound and so we look at we'll focus on this part a little more to see what's What's going on? Forgot the important point here
06:23
Francis Crick had Noted early published in JMB, you know that famous journal that everybody wants to push in these days He says It's just tempting to wonder whether the primitive ribosome could have been made entirely out of RNA and
06:45
That makes sense because at the beginning How could How could anything what's something other than the protein make the first protein? hence RNA so
07:01
Following on Francis We looked into it and indeed I'm skipping over Many many many talks on how we got to this point. This is the The center of the catalytic site with the p-site substrate
07:23
here and the a-site substrate Here the alpha amino group poise to attack the carbonyl carbon Be oriented Entropy is the most important part of catalysis. So that's part of what the catalytic power is
07:43
But there are other things but it's not being done By Proteins on the ribosome Now just to get a picture of What the reaction is like putting together a lot of work
08:02
Over a period of time. Let's put together a number of states This this of course shows you how you make a movie whoops And so, okay now we'll see how it works ah
08:33
That's how it doesn't work All right
08:45
Okay, there's the ribosome 50s subunit the large subunit the proteins and Now we're going to hone in on the active site Coming. This is where all the action is going to happen and we have the
09:13
What's called the a site and the p-site? Okay That's the p-loop that's the a-loop and now coming in this is an analog of the
09:28
p-site substrate There's the mean away so CCA Okay
09:40
Now we have the attacking the attacking a site substrate coming in and The alpha-meter groups going to tack the carbonyl carbon, but the structure isn't exactly right So there's a conformational change to orient
10:00
attacking the alpha-meter group to attack the carbonyl carbon and Then we're going to have an attack Coming up shortly. It's almost about to happen It's about to happen it worked now we have a tetrahedral carbon
10:28
and We have the product now we have a peptidyl group on the on the a site substrate
10:40
And then we have to have to change The p-site substrate leaves and and wanders over to the e site the exit site and it can only
11:03
the de-isolated tRNA or in this case analog can bind to the This side because there's not space here if there's an amino acid on so it's a way of moving the tRNAs Along the route to the p-site and now the
11:26
This the substrate in the a site has to go to the p-site so we can start this process All over again and there we are okay, that is by far the most popular movie
11:59
And it's it's great, okay
12:03
The rest of my movies don't have sound unfortunately Martin Schmang Wonderful graduate students. He's now on the faculty in Canada. I always told him that if he didn't get tenure he had a possibility of making movies so
12:22
Okay, so now I'll talk about a little bit more detail about the the mechanism a pro proposed proton wire Mechanism of peptide bond formation, and this is the reaction that we've just been through I have The first step is the attack of the alpha-immuno group on the carbon-carbon
12:47
Rise to products We saw got the a high-resolution structure quite high The
13:00
Complex when the pre attacks complex here 2.7 axioms and post catalysis complex at 2.55 axioms and we can put it together to make a movie I can show a lot of more detail, but instead I'm just gonna make the movie just to show you
13:20
how complex it all is and by the way, this can only be done if you have high resolution x-ray structures And you have to have several states as I said and so now here we have the a site P site substrate
13:43
aligned so that one can have Pep the alpha-immuno group attack the carbonyl carbon and this is the complete active site and they're water molecules now and other side chains
14:00
Orienting everything in there. Yeah have important to see the water molecules. There's a magnesium ion So another water molecule all Surrounding this the substrate And then we're going to see what happens in the process
14:23
Great, let me a nucleophilic attack and protonation right here Okay See these this is what's happening Three protons are in flight so to speak
14:41
And then Okay, that's the first step and then or a second step and then we have the tetrahedral intermediate That's the tetrahedral intermediate and Then we get the next step
15:01
breakdown of the intermediate and we have What's going on? The water molecules are playing a role in this In the active site and facilitating their action so now we have the new peptide bond and so that's
15:25
That's it So So that's the reaction Now, let's talk about a couple of these factors. I put that in the title. So I have to do a little bit of that
15:41
and We'll talk about how EFG facilitates tRNA messenger RNA translocation The question is oops How is it that EFG results in the translocation here
16:03
most of the models that were shown early was the EFG just flew in and Translocated but of course it doesn't happen that way and so what was found by Various people is that there are different states
16:23
Of the reaction There's a pre-translocation intermediate and post Translocation and we saw the structures of the pre intermediate and post state
16:41
a couple of very good postdocs in the lab in this and so the in the pre-translocation state the EFG is in a compact form. You'll see it. It's very compact And then there's a swiveling of the head of the
17:04
30s you'll see in a movie And then It goes into the non-compact sort of pushing it out as you You'll see so there is there's the major conformational change and you can see it's
17:25
definitely able to move things so Here's the model of the translocation this goes from a non-rotated to a rotated state
17:41
You see small motions here and some motions here and then the Elongation factor G EFG comes in in this compact state
18:02
So it's very compact and there's the the P site substrate and the E site substrate now We've got to be able to get the E site substrate in Okay, so this Undergoes the EFG undergoes this conformational change and
18:21
Pushes it out opening up the a site Pushing pushing it off pushing it off. Okay fully extended And then it Then it rotates rotates back. Okay, that's the post state
18:45
So this step can be inhibited There are lots of different kinds of antibiotics that bind to the ribosome it's a major target of antibiotics And this is one Deteriomycin and it binds in this site here and if you look
19:08
Close in I you can see it In here and Move in on there. Oh there
19:22
There it is And it gets in the way of these conformational changes So there there are a number of ways in which antibiotics can work and one of them is just preventing the conformational change And then there's another factor that I didn't even know about when we started working on this. In fact, I'm not sure it was known
19:48
It's called Elongation factor 4 or EFG is another name for it and it The question has been and still is what's its what's its function?
20:04
Does it Prevent is it a back translocase does it just open up the active site and let out the substrates So here we have a structure of EF4 bound to the ribosome
20:30
There there's there's EF4 bound to the ribosome It's got this long long bit here and when a site see TRNA is bound it binds on top of it. Okay
20:43
Here's the messenger RNA by the way, and so then What is what is the EF4 doing? And bound to the ribosome Ribosome in a non-rotated state with this in here and if we close in on it
21:07
there is a tRNA That's where it normally would be the classical state and blue is what happens on the EF
21:20
4 is there And see what happens it gets The factor moves the tRNA Away moves it away by 20 angstroms so and There's a clock like rotation in the small subunit
21:48
so it rotates a bit and what that does is it opens up
22:02
The active site or the Yeah, so it opens it up Sort of pulling the CCA out of the out of the active site. So one one thing it could be doing is Just
22:22
Removing the tRNA and that's one of the hypotheses now there's several hypotheses that come up here But here is the EF4 and there's the EFG the question is what is the EF4 doing?
22:41
It could unlock the ribosome I'm going to skip through these rather rapidly And Stall peptide in the tunnel Or it could dislodge the a site tRNA would be another possibility
23:03
Or what it could be doing I would say at the moment not really understood Exactly what it's doing so So EF4 has a number of possible roles
23:25
And And EFG I've told you what it's doing it goes from its compact to its non-compact state Okay now in the last bit of time and it'll be really quick
23:43
What can we say about New antibiotics and how can we use this structure to design new antibiotics? here's a Antibiotic That I'd never heard about until we started working on this a couple years ago. It's a peptide
24:04
It's a small peptide. It's about I think it's 12 amino acids a bit longer than that And it fits in the tunnel so there's the tRNA and there's the tunnel there's the exit tunnel
24:23
And there it is Interacting with the side of the tunnel the thing that's unusual about this is it's in backwards So it's not just a peptide getting stuck in the tunnel It's a peptide that goes in the tunnel get sticks and binds backwards
24:42
Can you use it for an antibiotic? Don't know But it certainly is Strange there are a lot of strange things out there and here Here's where it binds it overlaps the binding site of
25:01
Small molecule antibiotics So the question is how do you make use of this information? to Get new antibiotics, and that's a long story, and I'm not going to go into all of it
25:24
I'm just going to say progress has been made so This is where the antibiotics bind on the scale of the ribosome is pretty small So lots of them that overlap and the general idea is to make use of knowledge of where
25:43
known antibiotics bind to design new ones by combining the existing ones or doing a completely new design Making use so this is we started a company some years ago Peter Moore and I
26:03
Call the ribex pharmaceuticals RNA x-ray crystallography of course ribex perfect, but then it was bought up by another company and the Greek owner
26:20
names his companies after Greek islands, so it's now called Melinta Therapeutics and one of these days. I'm going to find out where Melinta is and in Greece And they're using this information to design new antibiotics and in fact they now have
26:41
One in license compound that they have worked on working on 15 years and and one compound Based on our work that is now Completing phase three clinical trials and So the idea of this strategy behind all this is to look at
27:06
Antibiotics bond in close places The first compound which is now for days lid, which is now completing clinical trials Has completed phase three clinical trials is combining the these two compounds into a new compound
27:29
And then there's another one Combining these two and then looking at the whole whole region and that's now Getting to what we call rxo4 and that's what everybody's really excited about because they're
27:47
Completely different antibiotics, and there's several candidates that are coming up So I think the structure based drug design game is going to work very well And I'll just quickly mention the first
28:03
Antibiotic the one that's now completing phase three clinical trials So this is this is what the precursor
28:25
Looks like bound and This is the minimum inhibitory concentration And anything that's
28:42
In No, I knew that was wrong That's our compound the the nasolid starter compound this is this is
29:00
Its effectiveness against a number of different strains MRSA for example terrible 64 influence a terrible terrible terrible If you look at the combined one this is this is the Linase lid or sorry the radies lid
29:21
Anais lid goes to radies lid You can see everything's in green So it works, okay, and of course you have to make sure that they're The Effective in and not causing any side effects
29:44
Which so far seems to be correct? And again, just just checking on it. Here's his radies lid versus linase lid against many different Varieties of ice let's
30:03
Linase lid which is sold by Pfizer Has its challenges Most of these are not effective Whereas for days, would they all work? so And this is the the final take-home lesson that I'd like to make
30:24
That is that Basic research Not only leads to understanding of biological processes that we all find really really fascinating and interesting oven by themselves in
30:41
This case it can provide information that can yield Compounds that are effective against all resistant strains And so I'm I have great hopes for the future of this kind of approach and and solving these problems
31:01
And here's the list of the people who've worked on it over the years Collaboration with ribex pharmaceuticals. This is just a few of the people there Peter Moore Collaborated and then I've had a number of people over the years who've been involved in this and I'll stop there. Thank you