Ada Yonath (2016) - What was First, the Genetic Code or Its Products?

Good morning. I prepared a different starting way, because I thought that I have to explain why I talk about a title that has more biochemical, biological meaning than physics. But actually I don’t think I have to do it now. Because I see that most of you are interested in such titles, in such projects. And second, our project was really dealing with biological, medical problems. The method was physics, crystallography. And the result is in chemistry. So maybe I convinced you that it belongs here. So the genetic code, I am sure you know what it is. I was younger than you, but not much younger, when it was found or when it was established. But now everybody knows, and even children know. When I talk to young children, 6, 7 years old, and I ask them, "You know what is the genetic code?" They say, "Of course, this is the tool that the police is using to identify criminals." (laughter) So we will not talk about each gene on its own. Most of the genes that any organism has are common to the same organisms of the same type. Some are different, like blond and black and so on, but in principle. The genetic code is the code that decides how we work, how we live, what we are or what any other organism is. And its products are proteins. And proteins are the workers of every living cell, from bacteria, cockroaches, flowers and so on. So I don’t think I have to tell you what proteins are or what they are doing. But if you want I can, in the intermission, talk about this. But I want to show you the DNA, the DNA that all of you know now its structure. It's a double helix that is connected, the 2 sides of it are connected by pairs of bases. The bases are the DNA language. There are 4 of them, that’s all. I am until now, since I was a high school kid, I am impressed by the fact that all life on earth is coded by 4 letters. So these 4 letters are the clue how to put together the amino acids that are making the proteins. So the DNA is 4 letters, proteins of 20. Proteins are long chains, I think you know all that. And the way that the flow of the information is shown here. This is the DNA as you saw earlier, here. These are the letters, the bases for those of you that still don’t know that. Here the information in the DNA is not available, it’s covered by the structure. So it’s been transcribed into a similar molecule that has the great ability to live as a single helix. This is called RNA, in this particular case messenger RNA. This is done by transcription, which in my drawing is just an arrow but actually it’s a very, very complicated process. And Roger Kornberg got for it the Nobel Prize in 2005. So then the messenger RNA which has again 4 letters, 3 of them exactly like DNA. One of them is slightly different but chemically it’s not important. It’s been translated to growing proteins by ribosomes. Ribosomes are cell components that know to translate. So now that we know how they work, we love to describe them as factories. Like this factory that is making a long chain. According to the code it comes in linearly. The factory is made of 2 floors. The top floor gets the code and decodes it. And when there is here a triplet - and this I didn’t say yet: each triplet of the combinations of the 4 letters is coding a specific, a cognate amino acid. So the amino acids are being brought into this factory on trucks by another molecule called tRNA. All tRNAs are very similar, but each of them is specific to its cognate amino acid. So they are being brought here. And when a triplet that is coding for this amino acid is found, the amino acid will get in and connect to the newly grown protein. Now this happens inside. My arrow is inside, but I don’t know how to draw inside, so please forgive me. And then the truck will be emptied and go out with the empty trucks. And this elongation cycle that I just showed needs some energy which I symbolise by these chimneys. But not much, it’s very efficient. So that’s the story. This is the way ribosome works. Just to go from the factory to life. Ribosomes are made of 2 subunits, small and large. The small is where the decoding happens. Here is the messenger RNA coming in. And the decoding is using the tRNAs. The tRNAs, each of them has an anti-codon loop that can make the same base pairs And there are 3 sites, each of them a triplet. Here in the P-site, peptidyl transferase centre, is where the tRNA will be connected to the next tRNA in order to make the chain. And the protein chain comes out of the ribosome through a tunnel that spans the large subunit. Once this bond is being made, everything will move. An aminoacylated tRNA will go into the P-site. Now it sits here and waits for the next tRNA. And this empty one will come out through the exit site. And that’s all, so simple. Took us only 20 years minus 2 weeks. (laughter). It’s important these 2 weeks. So we made a movie but, unfortunately, I can’t show it to you. So I’ll show you the movie in the movie file - hopefully it works. I think it’s important. So actually what this shows is what I just described: Messenger RNA is coming to the small subunit. So in the cell the 2 subunits are separated, each of them is an entity on its own right. They associate together when they have to start. And the starting is when the cell meets. And also it’s activated by initiation factors. So in bacteria, that’s what you will see here, the movie is about ribosomes of bacteria. The initiation machinery involves 3 factors, 1, 2 and 3, and you will see number 3. Maybe you can already see the end of it, you will see it in a minute. In higher organisms the initiation is much, much more complicated. But the ribosomes are also larger and more developed with evolution. But the main functions of the ribosome, the decoding and the production of the protein making the peptide bond, are done the same in all ribosomes, no matter where they come from, the smallest and the largest. So I am showing here the smallest, the bacterial. This is what we had 13 years ago when we did the movie. I want to tell you the movie exists in youtube - you can take it if you like or look at it - for 13 years. And so far there was nobody that complained. You think that this shows that they are correct or at least as correct as possible today? I think so. Anyway, messenger comes to the small subunit. And the small subunit takes it, look now, chuck, chuck, chuck, puck. Did you see the puck? The initiation factor number 3 is sitting here and monitors it all. And once it’s happy, the first tRNA is brought by initiation factor number 2. When everything is now monitored and happy, the large subunit can come and associate with the small subunit by surface complementarity. And by bridges that are made chemically between the 2 subunits in bacteria, 13 of them. Have a look, you see these bridges. So now this is the association of the active ribosome. Once it's active, tRNAs can be brought into it by factors that are called elongation factors. They are being decoded here and the peptide bond is being made here. And you see the ribosome is really happy and really helping, in/out, in/out real quick. I didn’t talk to you about speed but it can make up to 40 bonds a second, in one second, with a mistake rate of 1 to 1,000,000. I was a good student in second year, really good, and I got a really good book, how to make a peptide bond, the "Berichte". It took me 6 hours, I was the fastest. So they can make here, these are the peptide bonds, they can make 40 in a second. So you see here they are making. But I want you to pay attention to something really important. And this is that the main motion of the tRNA is sideways, whereas the lower part is rotating. So this is pseudo-symmetrical to this. There is in the lower part, which is the only part of the tRNA which is a single chain, there is rotation. And I’ll come back to it in a minute. But you see it comes in like that and goes out like this. And hopefully I can show it to you, yah. The tunnel is within the large subunit. Now you can see it here, this shiny thing. And the protein comes out at the end of it. The tunnel protects the newly born protein. It does other things too. And the protein folds when it comes out until there is the end of the code. And then instead of tRNA there are factors - recycling factors, release factors – that replace the tRNA and everything dissociates. And can go on to the next protein or to whatever the cell needs. So this is the protein coming out already folded. And here are all the players in this game that are now ready for the next task. So let’s go back now to the talk. In the movie the ribosomes looked very fuzzy but actually we determined the position of each and every atom – in bacteria a quarter of a million atoms. You can see here the small and the large subunit rotating. Each of them is made of many components. Ribosomal RNA is the grey, is the main one. And many ribosomal proteins in many colours. Any question here, no? So please don’t disturb. So the small subunit makes the decoding, the large makes the nascent protein. So when we look at the sites, at the regions where the activity is, in the large and in the small subunit, we see that here is the decoding. Here is the production of the peptide bond. And they are connected by the tRNA that always have this L-shape, double helical L-shape with anti-codon loop here. And the end of it, what we saw in the movie, the end always single chain, always fully conserve for evolution, CCA, cystosine-cystosine-adenine. So it is being decoded here. And this part reaches here. And here, where peptide bond is being made, and where the decoding is being performed, it’s only RNA, only ribosomal RNA. This means that the ribosome is actually a ribozyme, RNA enzyme. And when I asked these young children, Why do you think it is proteins can do almost everything that the cells want? They could have done it themselves. Why does the RNA make them? You know what these children answered me, these ones that talked about the police? They said, Because they don’t want the bias. If the cell had proteins making proteins they could have bias to the ones they love, the ones they don’t love. So this is the way the cell works and I think that the children are right. It’s a very sophisticated regulatory machinery. It is also in good agreement with the composition of ribosomes, I just showed you. They are mainly made of RNA. And it is in agreement with the idea that before life there was an RNA-dominated prebiotic world. So there are people that call it RNA world, maybe there were more elements there. I don’t want to go into it because I am not studying it, I am not an expert in it. But it was mainly RNA. And another thing is, RNA when they become enzymes, in our days they are really, really bad, lousy, lazy, whatever you want to call them. They can become efficient, like the ribosome is. So nature has mechanisms that are beyond our understanding as yet. So when we looked at the structures and when we look at the way the RNA is arranged in the ribosome here. You see the RNA of the large subunit and the little local double helixes that it makes and non-double helixes structures. When we look at it we don’t see any symmetry. We don’t see even any pseudo-symmetry. And the tRNA, if you remember, came out in a pseudo 2-fold rotation, I showed it to you this way. All of it moved sideways and the lower part this way. So there is no symmetry here and no symmetry in the sequence of the nucleotides. But if we look at the structure, this part has internal pseudo-symmetry like that. So this part can be rotated around this and come out to this part. Only the chain, not the sequence, not the nucleotide. I want to show it to you in larger, in higher detail. So this part of this region, the top part, is accommodating the P-site tRNA, the end of it. And the lower part is accommodating the A tRNA. And altogether it's 180 nucleotides. So if you want to see mathematically: we put the main chain of this on top of this or this on top of this by 180 degrees rotation. And we got that. You see how similar they are? They are not exact, of course, because one of these sites is accommodating tRNA, the other one is getting rid of it. But they are very, very similar. In the ribosome they sit here in the centre of activities. It is directly connected to the 2 hands. You remember in the movie, in/out. So it’s directly connected chemically to the 2 hands. And for the tRNA in one of the bridges it’s connected to the messenger RNA of the small sub unit. What you see here is only the large one and the position of this region. The blue and the green I will keep it blue and green. Blue, like here, for the A-region and green for the P-region. So this is the place it sits. And here is the position where the peptide bond is being made. It means it’s in the centre of everything, of activities. And we think that it can be a useful signal transaction because messenger RNA has to move here by a triplet when a peptide bond is being made. But how will it know it, it’s so far away? And how will the A-site tRNA know to come in when the peptide bond is made and the other one went out? So we think that this can be transmission of the signals. I want to show you the size of it. Because in this picture we focused on it but it’s, of course, not the exact one. So what you see here rotating is the large subunit. And this is the size of it, that’s all, this little piece. About 3 to 4% of the structure. But anyway that’s all. This is the way it looks for the chemists here or those that are interested. Grey is the ribosome and the tip of tRNA, A and P, are shown here. Peptide bond will be made here. And in order to show it more clearly: I have down there again the ribosome in grey, A and P blue and green, and peptide bond will be made here. That’s it. And this is the CCA, the conserved piece. If you want to see it, the machine, this machinery that we look at here. From the side it looks like this, the blue and the green. And here the CCA of the A and of the P. So as I said it’s the same in all ribosomes. In every ribosome, bacteria, elephant, dinosaurs, everything. So if we just look at this region, that we discussed earlier. I superposed all the known structures 6 years ago or something, there are here several. A is the blue and green is the P, as before. Here is peptide bond formation. Look they are the same. Once I superposed, I cannot even separate them. Of course, I superposed only the main chain. And here I put in the rotation axis, the imaginary rotation axis, to show where peptide bond is being made. So the high conservation of the symmetrical region or semi-symmetrical region indicates that its existence is beyond environmental conditions. So we called it the proto-ribosomes. We proposed that this is the beginning of the ribosome. That’s the way it started. So what does it say? That the prebiotic bonding entity, termed by us the proto-ribosome, is still functioning in the contemporary ribosomes. In us, all the time, millions of them. You know how many ribosomes there are in cells? In the liver they can reach 4 to 5 million. And in every other cell thousands, hundred thousands. So this is what we say. And if I write it in a prebiotic way, old letters. There is an RNA apparatus with catalytic capabilities that is functioning within the contemporary ribosomes, which means in all ribosomes. So just to show you again: it looks like that, I enlarged one of the previous ones. And what is how you here is crystallography result for the A-site, and mathematically derived rotational for the P-site. CCA of the tRNA. So we have an idea, we have many hypotheses how it came about. And one of them is the dimer hypothesis which we like very much, but we may find others too. So there were dimers like this in the prebiotic world of RNA that could self-fold, self-associate in a way that could make a pocket and make inside reactions. So it was actually a dimer. And I must say that we were very alone in the field for a long time. Until a group in Montreal showed it in a different way. So now it is more acceptable. So we are trying to make these dimers. We either make RNA that should fold similar to what it is in the pocket, which is stem, elbow, stem. Or we take it from what is available in nature, this part. So we can make these type of dimers, this type of dimers. These are the parts that are in the dimer all together, A and P - the positions of them in the A-site and the P-site. What did we find? Something, a very highly surprising result. There is a non-uniform tendency to dimerise. Not all of our constructs dimerised. Between A and P, only P. And between others about half of them. So we don’t really understand what’s going on but we think that we discovered the pre-Darwinian Darwinism, that even molecules can decide if they want to go on or not. So based on the suggested existence of an RNA-dominated world. And on the finding that RNA can replicate and elongate itself and has catalytic capabilities. This is all what we base on. We propose that the proto-ribosome is the entity around which life has evolved, not only ribosomes. So now we can try to answer the question, What was first - the genetic code or its products? Which is what I asked in the beginning. Or we can discuss for a second the emergence of the genetic code. And it’s just a possible pathway, it doesn’t have to be THE pathway. So possible substrates are shown here. Neither of them is ours. Many good chemists showed it could be substrate in the prebiotic world. And we take it and we think that the initial dipeptides that were made in this machinery could be the substrates for the formation of the next peptide bond and so on. So in this way oligopeptides could be formed - not very long but there you go. And the existence of well-performed oligopeptides catalysing fundamental reactions, or stabilising the machine producing them, may have led to the emergence of the genetic code. Because they were needed. I want to show you 2 examples. This is an example of an oligopeptide that has lots of histidines that can carry metals. And this is the example of where the machine could be stabilised, which is still not bound there. So this suggests that the genetic code was created by, or according to, its products which were found fit and useful and therefore survived. And led to the creation of a primitive original genetic code which co-evolved together with the products. So I hope that I gave some idea and I want to emphasise: the genetic code co-evolved together with its ribosome and its products. So one little comment from inefficient RNA enzymes, I said earlier the ribosomes are lazy or lousy as you want to call them. The contemporary ribosome is very efficient. It shows that nature has machinery to make it efficient. And we think that this is the ribosomal proteins that you could see in the beginning. But here are 2 representations of all ribosomal proteins in bacteria. They all have very long extensions of internal loops, otherwise they are small. And they are stabilising the structure. So I focus here on 4 of them. And I show them here in the small subunit. And you can see how beautifully they enter the structure and stabilise it. I can't talk more about it but we have wonderful examples. So our hypothesis is based on the existence of self-replicating RNA molecules with catalytic capabilities and on the assumption that the genetic code followed its products. Just to show it where it is again to remind you. Here it is in the ribosome. In this very complex structure we identified this piece - this is here - and it amounts to 3 to 4% of the RNA in ribosomes. So during the 4 days that I have been here, everybody asked me about antibiotics - especially the media but also many of you. So I decided to give a few minutes to antibiotics. Over 40% of the useful antibiotics are hampering protein biosynthesis, mainly by paralysing the ribosome. Because it’s clear if the next generation will have less proteins or wrong proteins, it's death to the bacteria. Natural antibiotics are the main antibiotics we are using now. And they are the weapons that bacteria from one type is using to interfere with the cell life of different species. So the question is: antibiotics are very small, around 500, sometimes they reach 1,000; bacterial ribosomes are 2,500,000 - how does this work? So if we think about the factory that I showed in the beginning. Factory works in assembly lines. If you stop one assembly line the whole thing is done. So this is what the antibiotics do: they stop one function. And you can see here the shape of the large and the small subunit. And all these balloons, each of them is describing a family of antibiotics. Just a second for "What is a family of antibiotics?" It’s what the companies made from the natural antibiotics, they made them better. And they are all in the decoding, in the hinging, tunnelling and bonding. So now we have another movie that doesn’t go and maybe I’ll show it at the end. Because I see that my share is already up and I didn’t really finish. So we talk about antibiotics a few more minutes. All antibiotics bind to functional sites. And it’s based on very small differences - I won’t show it now. But the problem is what everybody talks about is resistance. And a prominent mechanism for antibiotics to acquire resistance is to change their own binding pocket. And maybe there is no way against it, but maybe there is a little bit. So I’m not sure that it’s possible to combat resistance fully. Because bacteria want to live, they were on earth before us, they are with us. But it was found that even in tribes that never had European food or medicine, there are already resistance mechanisms. So it suggests that bacteria knows what to do. What do we do in order to contribute to this? Until recently all the structures of ribosomes and antibiotics that were known were models of pathogens. That explained exactly how antibiotics bind, but not the species specificity of resistance. And they are different, they are species specificity in resistance. So we grew a ribosome from a pathogen. And we learned that it’s possible to make better antibiotics. You can see here a family of antibiotics that was improved just by adding one hydrogen bond - 16-fold higher potency. Second, beyond our expectations we found that some of the pathogenic ribosomes in pathogenic bacteria have insertions. So if we look at protein L3 in many, many bacteria, only here there are insertions. And these 2 are in pathogens. So when we superposed the ribosome and the structure of a protein, not the particular one before, but the latter one, of 4 different bacteria, only this one came out. And this is a pathogen. So we think that it can be a potential new species. The same is on the surface. All these cyan are extensions of the pathogen. And if we look at it, it looks like this. Everything exactly the same between pathogens and normal. But this is an addition. We think that we can use this as a potential binding site that the bacteria didn’t find out yet about. That the bacteria didn’t find out yet about. So we identified 25 new potentials like this. Blocking 16 really stopped the ribosome work because these positions are used for the ribosome to interact with other components. And we think that we can make better antibiotics. And here I come to environmental and ecological considerations. Almost all known antibiotics until now have cores that are not digestible, not degradable. So they contaminate the environment. And they come back to us for the grass that is grown on the contaminated water. So the newly identified potential binding sites can be exploited for the design of degradable antibiotics. As I showed earlier we already did some degradable - for the chemists: PNA, DNA and small amino acids, small peptides. So the insight we have can really help both antibiotic resistance and ecological. And I am not going to show it all, but they will help to separate between what we call good bacteria, the microbiome, the trillions of bacteria that live in us and contribute to our good life, and the pathogens. Because we suggest to make pathogen-specific antibiotics, for each pathogen its own antibiotic. So this will also reduce resistance. So let’s just say that this is a revolution in the antibiotic field that today prefers a broad range. For having this we have to identify what type of bacteria. And we have to find a smooth way to find out the important, unique features of each pathogen. Both take a long time - look at the time limit. The first structure, 20 years as I said earlier. Afterwards there were structures that came out at 5, 3, 4 years. This is not good, we want to do structures in months. And for this there is now a new way, cryo-electron microscopy. And I just want to show you that we did solve several structures with it All the RNA, all the proteins and for the crystallographers, the level of accuracy. It’s not always like this but in this structure. And we can even see mutations. So thank you really very much.(Applause)

Ada Yonath (2016)

What was First, the Genetic Code or Its Products?

Ada Yonath (2016)

What was First, the Genetic Code or Its Products?

Abstract

Ribosomes, the universal cellular machines for translation of the genetic code into the cellular workers, namely the proteins, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases. The site for the creation of the newly born proteins is located within a universally conserved internal semi-symmetrical region. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent a remnant of an ancient, prebiotic RNA machine. Hence, it could be the kernel around which life originated. The mechanistic and genetic applications of this finding will be discussed.

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