Ada E.  Yonath (2013) - Curiosity and its Fruits: From Basic Science to Advanced Medicine

Good morning. Thanks for coming to listen to what I can tell you about ribosomes. I take it for granted that you know something about the translation of the genetic code. And this was my, the point that I really wanted to understand, the point of curiosity. So actually what we did is basic science, curious basic science. But I was really lucky and within our work we also could find implication that has to do with advanced medicine. And we are very pleased about it. Although I didn’t even dream that it will be possible. So let’s go into it. I am sure that you know what proteins are. But I think that not all the chemists are well into proteins. I just say that these are the cell components that are facilitating or performing almost all cellular functions. Those required for smooth life and those required for pleasure, for quick, fast, first reply to problems. And some problems are on the wall. I also think that you all know that in science, in chemistry, in nature structure means function. It means it’s not enough to have the material or the compound itself. And I use here paper clips as the compound. They also have to have the right structure, the right fold, the right conformation. So paper clips here can put together several pages and keep them together. And with my own fingers I changed their fold. I didn’t dissolve them, I didn’t cut them, I didn’t do anything to them, just unfolded them to this type or to this type. They are the same, everything is the same, the chemistry is the same, but not the fold. These cannot do their job. So we need in nature, in general we need to keep the right fold. It means the materials, the compounds, those that are doing the jobs have to have the right fold. And this is correct also for proteins. Proteins are long chains made of amino acid. And the fold of each protein, which is carefully designed for fulfilling its function, is determined by the sequence of the amino acids building it. There are 20 types of amino acids, I’m sure you know that. Their sequence within the protein which enables their functionality is dictated by the sequence of the genes, that code for the protein. And the genes are stored in DNA which is the string of instructions. They’re worth nothing unless they are translated. It’s written in a 4 letter language called bases in which each 3 of them code for a specific protein called codons. And please forgive me - for those that are, this was too simple. When we get to the ribosome we’ll be a bit more complicated. So DNA is shown here schematically. These are the bases. There are 4 of them and they are shown here, each of them is represented by a different colour. You have the names here if you like. And the instructions, these are the instructions. Each 3 of them will code for 1 amino acid but the instructions are not available here. Because they are masked by the double helical structure and by the packing. So first thing that has to be done: The DNA has to be transcribed to messenger RNA, to RNA which is almost the same like the DNA but it doesn’t have the deoxyribonucleic acid. It is just ribonucleic acid. It has 4 bases, 3 of them are exactly like in DNA and 1 is a little bit different - I won’t go into the chemistry now. The transcription is a very complicated process. Here it looks like a little box but actually it is made of many steps. And Professor Roger Kornberg got the Nobel Prize for unravelling them in 2006. The messenger RNA - that is the product of the transcription of the piece of the DNA that has to be translated And this is what we wanted to understand, how this happens. It was known, when we started the whole thing was known, but not how it is done, how it is performed at the molecular level. So we like to call the ribosomes, the translators, we like to call them the factory of the cell. A universal factory that exists in all living cells, that can function almost like a factory with an assembly line. So the instructions are coming into the ribosome linearly. This is the messenger RNA. They come in, they are being read here. Some energy is needed for it but I think not exceptionally high, actually very efficiently. They are being read here. tRNA which are other RNA molecules that can carry amino acids according to the code are bringing them. So we drew them as trucks. Each amino acid here has a different colour. This shows that it’s a different amino acid. Now this truck will come in. This amino acid will be stuck to the growing protein. Of course this happens inside. But I don’t know how to draw inside, so I drew it here. Well we don’t know everything, at least not to DO everything. And now the protein will grow by 1 amino acid. The trucks can go out and look for another job. And the whole elongation cycle needs 2 GTPs and this is correct for all ribosomes in all cells: from bacteria to elephant, fish, plants, whatever, cockroaches - whatever you want. So ribosomes are really clever. They can read instructions in a language of 4 letters. And they produce their product in a language of 20 letters, the 20 amino acids. They are universal. All ribosomes work the same way, I already hinted it. Perhaps exactly the same way but, if not, very similar. And this is regardless of their source. Ribosomes gained mass with evolution. So the ribosomes in bacteria are smaller than ribosomes in human but the activity is the same, the functionality is the same. Although they are very, very large compared to cell components there is a huge number of them. And they function in each living cell, mammalian cells can contain millions of them, especially in the liver. And bacteria can reach 100,000 in their growth phase. They act continuously. They can form, chemists listen, 5 to 40 peptide bonds in a second. Anybody of you made a peptide bond in the lab? You’re laughing - I did. Second year this was our exercise. It took me 6 hours for 1 bond - not 5 to 40 in 1 second. I hear that now this reaction has progressed and there is some better equipment. So it takes 2 hours to make 1 bond. And mistakes are allowed according to the chemical books. But if the ribosome does it what we can catch is 1 to 1,000,000. I am sure that they make more mistakes but they have a very good proof reading machinery that kicks out the mistakes. So what we can measure as human beings is between 1 to 10,000 to 1 to 1,000,000. They all are made of 2 subunits, small and large. You remember, in the factory the instructions were in the top floor, in the small. And the peptide bond is made in the large subunit. The amino acids are brought by tRNA. tRNA are universal molecules, very, very similar to each other. All have an anticodon loop and carry the amino acid that is connected to it on the other side. So here is the decoding, here is the decoding happening. Actually here is the point of decoding. It’s called the P-site, peptide transfer site. The site next to it where this new tRNA will come in, is called A aminoacylated tRNA. And once a bond will be made this one will be empty. So it can go out through the E-site, which is neutral empty and also for exit. That’s the story. Most of it was known before but not 'how'. The question of sites and so on was not so clear. Pay attention, each site has part in the small subunit and part in the large subunit. Peptide bond is being made here in the large subunit. And the newly born protein emerges out of the ribosome through a tunnel that protects it as many other things So instead of talking, I will show you a movie. A clip that we made 12 years ago when these structures came. We interpolated several structures into it, not only ours. Each structure captured the ribosome under different functional states, so we could interpolate. The interpolation was done sometimes easily and sometimes we had to think. So, for instance, you will see a motion like this. It is an interpolation between this structure and this structure. And I had the guts to say it goes this way and not for instance this way until it reaches. Because I thought that nature is at least as clever as I am, probably more. And later, after a few years, the in between structures were also determined. So now we know it’s right. Those of you that fall in love with the movie, YouTube has it. So the messenger RNA reaches the small subunit. And it takes it, look, look, look now, - tshup: took it. It means this little motion from here to here which looks very little. It’s engineered exactly to let messenger go in and not go out and not move. And the part that has to have the dynamics for it, is almost quarter of a million daltons. It’s not a joke. But in the movie it looks little. There are factors, non-ribosomal factors, that tell the ribosome "start". They’re called initiation factors. And here what you see is the initiation factor number 3. We know exactly where it is crystallographically. And it will leave together with initiation number 2 that you will see in a minute. And 1 I won’t show today. So the factors, 1 is here and now, it makes sure so, it makes sure that the messenger is bound correctly. And that the codon that has to be first is exactly in the P site. And now comes the first tRNA brought by initiation factor number 2. And being positioned exactly in its place - if it’s not exact it won’t work. So what we have now here is the initiation complex. Once the initiation complex is produced, the large subunit can come. Associate according to surface complementary but also make bridges, conformational changes. Now tRNAs can come. They stand here and come in according to the need, according to the gene. In out, in out, the ribosome really helps - you see the 2 hands - and in and out. Don’t laugh, it’s more than a 100 nucleotides and a large protein. And here 4 proteins. So in out, in out, 40 times a second in bacteria. And the protein is being made here. We took away the large subunit so you can see how the protein is made. I really won’t talk about details. But you can see the main motion of tRNA and also some changes in the motion. You can see that the protein goes out through a tunnel, a tunnel in the large subunit. Now the protein is long enough to reach out of the tunnel. It comes out, it exits. It should fold correctly alone or with chaperons. And the process continues until there is a stop codon that tells "stop, end of protein". And instead of tRNA there are factors that come, like release factor, recycling factors, this gold thing that comes in separates the 2 subunits. Now they are dissociated. Protein can go, do what it needs - you see it. And the tRNAs can look for a new job. That’s the whole story. Took 20 years of our scientific life. Applause. Thanks, thanks, but I still have to tell you something. We made the movie together with 2 art students in the art academy in Jerusalem. So I will tell them that you liked it. And let's go on. So actually in the movie you couldn’t see details, but in life we see we can position every single atom in the small subunit which you see rotating now. The small subunit that you see rotating is from a bacteria, thermos thermophilus. Its sediment is 30S. You can see here some chemistry of it if you want. And it does this to decode the genetic code. What I want you to pay attention, that the colour that is dominating is sort of white or light blue in here. And this is ribosomal RNA, so it’s another RNA molecule. And there are many proteins, up to 21, in the small subunit, each of them in a different colour. This structure was determined in parallel but not together. It was made in the lab of Venki Ramakrishnan and the 2 structures are exactly the same, so must be correct. Actually the whole thing must be correct because the movie was never objected. Now the last subunit, the same story: mainly RNA, ribosomal RNA, sediment is 50S. A task is to make the peptide bond but not only 1, to elongate it, polymerase. It is of much higher molecular weight, 1.5 megadalton in bacteria, 3,000 nucleotides and up to 34 different proteins. So now you can see that we see essentially all atoms. Let’s see how protein is being made, how the peptide bond is being made. So we have here a view into the PTC, peptidyl transferase centre. That’s the active site and it’s in the large subunit. You see here the 2 tRNAs and the components of the peptide bond that will be made here. Around it in grey is the ribosome, only RNA. And what it does, it really positions the 2 tRNAs in stereochemistry that allows for peptide bond formation, c'est tout. And if you want to see the 2 tRNAs in space filling, you can see them here. Again the ribosome is in grey. I will use the blue and green for A and P site tRNA in the next slide. So I already hinted at it. But when we look at the whole surface that will come together in the whole, in the entire ribosome And the active sites, the decoding and the peptide bond formation. You can see them here and here. They are just in RNA. No protein in altogether. So they will come up together like that. And tRNA which has an anticodon loop that will associate here. And another piece, double helical piece, that is called acceptor stem, with a universal, fully universal 3 prime NCCA amino acid will hit here. This is correct for all ribosomes, it doesn’t matter how large they are around here. So the ribosome is actually ribozyme namely an RNA machine. And what does it mean? So when I ask younger people, younger than you, much younger, like high school kids or even grammar school kids, and ask them "What do you think it means: RNA is the active site?", they say that the cell doesn’t trust proteins to make themselves. Which looks childish, but I think it’s correct. I think that this is a very sophisticated regulatory machinery that the cell provides. So RNA, one kingdom is making another kingdom. Not proteins making themselves. Because then they can be biased. You laugh, you see- It is also in good agreement with the composition of the ribosomes. You could see that most of the ribosomes are RNA. There is one exception in mitochondria that part of the RNA was replaced by proteins. But still the active sites are just solely RNA. And it is also in good agreement with the idea that there was an RNA well before proteins. So you see how basic we went even into this question. So we looked into, once we saw that the structure, the active site is RNA alone, we looked into it. And we found that in all known structures. It doesn’t matter if they are bacteria, eubacteria, archaea, eukaryotes, even mitochondria. There is an internal piece, internal region, that is semi symmetrical. It’s made of 2 sides that are almost symmetrical to each other but not exactly. And this region is the same in all ribosomes from the point of view of sequence. And what we know now of structure - I have here several structures that are known today And I give my head that you don’t see which one is where, they are really one on top of the other. And semi-symmetrical because the reaction is semi-symmetrical. A and P have to come like this. You have seen it a minute ago, it will come back. So actually what we found out is that the ribosome architecture hints at its origin - or that’s what we think. Within this complex structure, you saw how complex, we identified a highly conserved, semi-symmetrical region around the ribosome active site. That can be a remnant of prebiotic bonding machinery, bonding apparatus Then we call it the proto-ribosome. This region is about 3 to 4% of the whole ribosome, although it looks here much larger And it’s still functioning - if it’s correct it came from prebiotic time - still functioning in us. So you want to see the size? You see here the whole large subunit rotating. It’s not the whole ribosome, just large subunit. This is the size. So that’s what you saw earlier in space filling and in positioning. This is exactly this little piece here in the large subunit. And it has the structure, the whole structure is like that, and I increased it. Now you see the 2 parts of it with 2 substrates. Here there will be a peptide bond. And we think, this we think, we suggest that it existed and functioned before life and it’s still existing in the contemporary ribosome. You can see it here, this is the region. And it is directly connected to the 2 moving hands and the 2 tRNAs. So we think that this may be a centre of transmission of signals. And in support of it, a protein that was sitting here was kicked out in another lab, in another ribosome, and changes in the structure were found here. So there is a transport of signals between them. So the high conservation of the symmetrical region indicated that its existence is beyond environmental conditions. And this is called, in our world, the proto-ribosome, 'before ribosome'. So it’s a prebiotic bonding entity that it still functioning in the contemporary ribosome. I don’t know if I got you excited, but we are. And Miri Krupkin, she is here, she is working, she is trying now to prove it experimentally. And this is based - not only we want to call it a proto-ribosome - it’s based on the suggestion that there was an RNA world. And on the finding that RNA can replicate and elongate itself and can have catalytic capabilities. And as I said we think that the world was, life world was made around it. And this implies that the genetic code co-evolved together with the ribosome. As well with the products, the proteins. It means proteins were actually small proteins even before the genetic code. So maybe you want to answer the question now: What was first? I just hinted. The chicken or the egg? In chemistry: the genetic code or the proteins? Anybody volunteer? No volunteers? Egg, why, who laid the egg? So we think neither of them. The proto-ribosome was first. So until now I told you about basic science. Let’s go for a few minutes into advanced medicine because of the fundamental role played by the ribosome many antibiotics target it. Stopping the ribosome is not killing the ribosome, the ribosome is not alive. But it’s stopping any function of it, it’s like stopping in a factory, an assembly line, any step. This will stop protein biosynthesis. And the bacteria that will come out will be invalid. So this is the way antibiotics work. And indeed over 40% of the clinically useful antibiotics target the ribosome. What are antibiotics? The natural antibiotics are the weapons that bacteria from one species is using to interfere with cell life of another one. And the question is, "How do the tiny antibiotics paralyse the giant ribosome?" Ribosomes from bacteria are 2,500,000 dalton, Antibiotics are about 600 – 900 dalton. So how do they do it? Each of them binds to an active site, a functional site. And although they are small they can stop, they can stop protein biosynthesis. I will show you another movie. Since you liked the first one, maybe you like the second one too. It was made by us, together with the 2 students. And here Edeine antibiotic, look how small it is, but it sits exactly at the path of the messenger RNA, doesn’t let it go. Also minimises the mobility here. That’s it: no messenger, no protein. The second one is tetracycline. It is more useful but I don’t talk about clinical considerations now. It occupies the position of the second, the A-site tRNA. No binding, no protein. The third one is essentially the first anti-ribosome antibiotic that went into use, erythromycin, in the middle of the last century. Sits in the tunnel and blocks it, like a barrier. So a little protein can be made, 5, 7 peptide bonds, but no more. And the last that I’ll show you interferes with peptide bond formation. Just sits where the bond should have been made. Between the NH and the CO. Look here: hop, that's it. So very simple but very clever. And I just want to show you one, here is erythromycin entrance to the tunnel. This is the tunnel. You see here the whole large subunit. Protein will not pass. At least not more than polyglycine. So now there is a problem. All antibiotics bind to ribosomal functional sites. But they are highly conserved, I just said it. And mandatory for clinical use is the distinction between the bacteria and the patient. We want to kill the bacteria, not the patient. But if the site is the same it will bind to both. So luckily there are subtle differences. I want to show you a subtle difference. So what you see here is a section, perpendicular to the long axis of the tunnel. These are the tunnel walls in grey. Inside there are many, many structures. Each of them is an antibiotic that binds there. Each of them is represented, each of them was determined crystallographically. All block the tunnel better or worse, but all block. All interact with a position 2058 in E. coli which is adenine in all eubacteria, in all pathogen. But it’s not in others. So the contacts, the affinity, between erythromycin and A2058 is very high. Fantastic. In all eubacteria. And please pay attention to here, this is the difference between us and bacteria or bacteria in us. That’s it. G instead of A. From the point of view of base pairing, not important. From the point of view of space, available space, this is too close to here. So there is repulsion. No binding, tshik, that’s it. So now let’s go to a real big problem. There is very large resistance, very severe resistance to antibiotics. And the question is, it’s a very, very bad happening in medicine, and the question is: what are the mechanisms. So one mechanism is used A to G, G to A, for instance all other anchors. The other is allosteric. But the question that I really want to ask is: Can we combat antibiotic resistance? Do we have a vision to do it? So in my opinion only partially, because in everyday awareness it’s because bacteria want to live. They were on earth before us, with the dinosaurs and whatever. They are with us, they want to live and they will find a way. Still we can look at some ways to minimise or control resistance. And I think we should do this. And I want to show you one suggestion. Its synergism between 2 compounds. So again you look at the section through the tunnel wall, perpendicular to the long axis. It’s here in pink: one antibiotic, second antibiotic. The winning couple, isn’t it. One stops the other from going out and they block fully. So this is what happened to me when I heard about it about 12 years ago. And as a good chemist I wanted immediately to combine the 2 of them - why have 2 parts? Well I thought about doing it. Other people thought about doing it too. And they were faster and they failed faster. It means they just failed, I didn’t have to repeat. So why was the failure? Because the combined compound was much too large and didn’t find its way to the right position - and because of something else. So you remember this picture and now I highlight here one nucleotide. This something else we found only crystallographically, it was not clear. This 1 nucleotide is actually in the position of making the peptide bond. I hope that you remember this picture. What happens to it? So let’s look at it, this is the one. Without tRNA, without everything. And now we look at it from top, here. Here it is. Now compound A is coming in and kicks it out because it’s very flexible. And the compound wants to sit there. So it destroyed the active site. Life is very strong, so life will kick compound A out and bring the nucleotide back. But compound A is in surplus so it will come back. And if you want I can continue but I will not because compound B comes here and doesn’t let the nucleoid going back. And doesn’t let compound out, going out. So everything is locked. But it’s not just blocking, it is changing the active site, fully changing: 180 degrees flip So this triggered us to look for another pair. This is the pair that is in the literature and also sold. It is rather very poisoning and rather expensive. It’s not very much used but I just showed it to you. And we looked through another pair which is smaller and hopefully as good. It was found, it is made by a bacteria. It was found first in Sri Lanka and now in many others. And we want to, we show that it does the same change. That the 2 compounds interact very nicely. And we want to make it. So I have a dream before I finish and it’s called, I call it 'the blue dream'. This is the current situation, the 2008 situation of the whole world in terms of life expectancy: My dream is that soon the world will become blue. And for this companies have to work on resistance to antibiotics. And this is what I want to tell them. Before I finish I want to give credit to the Weizmann Institute that let me work on what was called 'the dream' for over 20 years: the president, the vice president and the scientific advisory committee. Everything started with this very strong collaboration with Max Planck in Berlin, with Doctor Wittmann, who died 10 years before the structures came out. So I had 2 research groups, one in Berlin, in Hamburg, that was responsible for the crystallography, taking the data, and one in Israel. I want to show you the one in Hamburg that terminated 9 years ago. That always had angels, the TAs for this too difficult project, that went to the Dead Sea to look for good bacteria - I can’t talk about it. And to show you the group in Israel that still exists. And I want to highlight Professor Raz Zarivach who is here too. Now he is professor in Ben-Gurion University. He was born in the ribosome. He traced almost all the RNA in less than half a year. And Miri who I mentioned, who wants to prove that ribosome was before us. I can’t talk about the rest but you can see. I only want to highlight Tamar. But before this: for the young girls here, the young females that hesitate, "Shall I stay in science or not? Because maybe science will make me a bad family person." I want to tell you, here you can see 3 scientists, fantastic scientists, each of them have 2, 3 children, and each of them work in science. Tamar not only has 3 children, she knows to bake, she made a cake for my birthday. You want to see the cake, look at it. So it shows that ribosomes for my group are considered sweet. I want to thank my family that supported me all the time emotionally, it’s my daughter that is an MD and my granddaughter that say in a speech she wrote about me: But she always finds time for me." Girls it’s possible, it’s possible to be a scientist and be family people. She gave me a prize - you know this prize here, you must know, it’s the Nobel medal. In my opinion she gave me a prize at least as good, maybe better: "The grandma of the year is Ada Yonath." Applause. And when I asked her, "Which year, the grandma of the year, which year?" do you know what she said? Ribosomes are really popular now in Israel, you can get the ribosome wig and become a ribosome. And I just want to tell you that my curiosity started when I was 5. I wanted to measure the distance from the floor to the ceiling. And fell into the back yard and broke my hand. So this is what happened to me. You see small subunit, large subunit, symmetry, and a new antibiotic that the artist thought about and thank you very much. Applause.

Ada E. Yonath (2013)

Curiosity and its Fruits: From Basic Science to Advanced Medicine

Ada E. Yonath (2013)

Curiosity and its Fruits: From Basic Science to Advanced Medicine

Abstract

Ribosomes, the universal cellular machines that translate the genetic code into proteins, are targeted by many antibiotics that paralyze them by binding to their functional sites. Antibiotics binding modes, inhibitory actions and synergism pathways have been determined for almost all ribosomal antibiotics. These indicated the principles of differentiation between patients and pathogens, suggested mechanisms leading to bacterial resistance and paved ways for improvement of existing antibiotics as well as for the design of advanced therapeutics capable of minimizing antibiotics resistance.

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