13_BL_20231103_WhisperAI_2.docx

Okay, and then finally, the last couple of minutes, I would like to introduce to you what ribosomes are. So the ribosome is the cellular machine responsible for protein synthesis. Ribosome is where all these critical interactions take place. So the interactions between codon and anticodons, the bind...

Okay, and then finally, the last couple of minutes, I would like to introduce to you what ribosomes are. So the ribosome is the cellular machine responsible for protein synthesis. Ribosome is where all these critical interactions take place. So the interactions between codon and anticodons, the binding of the messenger RNA, the exchange of tRNAs delivering different amino acids, this is all happening on a large cellular assembly called the ribosome. These particles are actually so large, they could be identified in electron microscope. So they're almost referred to as cellular organelles because they're so complex they can be seen, usually single proteins you would have difficulty seeing under electron microscope. But even in the cell, people could recognize ribosomes very early on. So for example, if they would look at the negative stain, electron micrograph of a cell, they could see certain membranes and next to them this pattern of dots. So Pallada discovered those and later they were called ribosomes. So what are these ribosomes? Architecturally speaking, ribosomes can be separated into two different subunits. So basically it means that under certain conditions if you would break open the cells and you would kind of separate the ribosomes, they're so large they can be separated from the rest of the proteins and small nucleic acids. And this is done similarly to the way I showed you. You can separate DNA molecules in a gradient upon fast centrifugation. Depending on the density, different things will sediment at different rates. So the same applies to ribosomes. So basically if you take these ribosomes, you separate them from the rest of the cellular components and you spin them down in a gradient, they will sediment at different rates. And this sedimentation, how fast things will spin, will depend on the shape of the molecule and on the molecular weight of the molecule. So larger things will sediment faster and deeper in these gradients. And so people realized that if they would isolate these ribosomes, depending on the conditions, if they would change a little bit the magnesium concentration, these ribosomes can come apart and then they could separate two parts of the ribosome. And they refer to those as large subunit and small subunit. So these two subunits are not only interesting in terms of architecture, they're actually functionally distinct. So large subunit has certain roles and the small subunit different roles. So the whole ribosome, you can think of it as consisting of two subunits like this. And in this view, this is called the crown view of the ribosome, because it has this little protuberances, stalks that are sticking up in this view. And then when you separate the two subunits, you have the large subunit with this protuberances sticking up, and the small subunit that has features that look a little bit like head and body. And together they come together and they form the entire ribosome. So how about this ribosome? Two subunits, different roles. Small subunit you will see interacts with RNA, with messenger RNA, and large subunit is the place where the newly synthesized protein is formed. It's the catalytic subunit. This is where the amino acids are brought together from individual amino acids into a polypeptide chain. The tRNAs as a consequence of this distribution of functions, where the small subunit interacts with messenger RNA and the large strings to get amino acids, the tRNAs obviously have to bind here between the small and the large ribosomal subunits. And finally, when the newly synthesized proteins are synthesized, they have to move through what is referred to as the tunnel. So it's a physical tunnel through the large subunit before the proteins can fold. And of course ribosomes can synthesize both soluble proteins and proteins that have to be targeted to membranes. So with this, I would like to conclude with the lecture for today. We will continue in the next 45-minute segment. And then you will hear more about the composition of ribosomes and the stages of translation. How the ribosomes find the start site on the RNA molecule, on the messenger RNA molecule. How they keep adding new amino acids according to the sequence and how they terminate protein synthesis. OK, thank you. Now, we will continue on the topic of translation. What you have heard about last time is that during translation codons in messenger RNA are translated such that a sequence of amino acids is covalently connected to produce a protein. The information in nucleic acids is encoded as codons, as triplets of nucleotides. And the correspondence between these triplets, all possible 64 different triplets, and what they mean in the language of proteins is referred to as the genetic code. You have heard that since there are 64 different combinations for triplets of nucleotides, there is many more combinations than necessary to encode 20 different amino acids. This is referred to as degeneracy, so genetic code is degenerate. Genetic code is also not non-random. That means that certain changes of these triplets typically do not imply a big change in the chemistry of amino acids that they encode. That means that certain types of amino acids, hydrophobic, hydrophilic, positively charged, negatively charged, tend to be groupated in this genetic code table that you have seen. You will also remember that some of these triplets, some of these codons, do not refer to a particular amino acid. Rather, they identify a stop, which means that when this codon is encountered, then the polymerization of a protein, the synthesis of a protein, is stopped. You have also heard that the enzymes that connect a particular tRNA molecule, which serves as an adapter between the codon in the messenger RNA and a particular amino acid, the enzymes that attach an amino acid to a specific tRNA, let's say, alanine amino acid is attached to alanine tRNA that has an anticodon that is capable of base pairing to the codon that specifies alanine in the messenger RNA, these enzymes that connect the alanine and the alanine tRNA are referred to as aminoacyl tRNA synthetases. These are highly specific enzymes, and there is 20 of them, and they will charge all different tRNAs with appropriate amino acids. You have also heard that since there is 64 different combinations for triplets of nucleotides, this is more than necessary, and because of that, the strict base pairing, Watson-Crick base pairing, is followed only in positions one and two. The third position is still checked, but not as strictly. That means that the Watson-Crick base pairing is enforced in first two positions, and in the third position, there can be some wobble. This is referred to as the wobble base pairing between codon and anticodons in the third position. The whole process of translation, therefore, needs a messenger RNA, needs an adapter molecule, tRNA, that interacts with messenger RNA, and needs a large cellular machine called the ribosome, where these interactions take place, and where amino acids are connected into a growing polypeptide chain. The ribosome serves to both control these codon-anticodon interactions and to catalyze so-called peptidyl transverse reaction that you will hear about today. Today I'll tell you more about ribosomes. You have heard a little bit how they look, that they have this crown-like appearance, that they have two subunits that have distinct roles, but you will hear much more about how the process of protein synthesis takes place on the ribosomes. Additionally, you will hear much more about what is referred to as stages of translation, just the way DNA replication or transcription took place in different stages. So there was a process or stage of initiation, elongation, and termination, and similar stages also exist when we talk about translation. So I introduced to you a little bit what ribosomes are, and how they look, what their architecture is, and today we will start discussing what they are composed of. So they're complex cellular assemblies, some of the largest, very well-structured, discrete cellular assemblies. They can be even seen in electron microscope images as cellular features, as little high-density dots. However, they are very well-defined, just like enzymes are, but they are order of magnitude larger than typical enzymes, like hemoglobin, or like lysosome, like some other enzymes that you might have heard about so far. So because of their large size, initially, ribosomes were characterized in terms of their sedimentation coefficient. Sedimentation coefficient is a physical characteristic of an object, of a molecular object, that is described in terms of how fast its sediments, under strong gravitational field that is generated during centrifugation. So at very high centrifugation speeds, at multiple times the g-force of Earth, you will end up sedimenting objects, and this sedimentation rate will depend on the molecular mass of the object, molecular weight of the object, and its shape. And different objects, again, depending on their shape and molecular weight, will sediment at different rates. And because of that, initially, bacterial ribosomes were characterized as 70s particles, because they were sedimenting at a rate of 70 Svedberg coefficients. So this Svedberg is the person who defined a coefficient that corresponds to a certain sedimentation rate. So bacterial ribosome sediments at 70 Svedberg when you separate the two subunits, you end up with small subunits that sediments at 30 S and large subunit that sediments at 50 S. Please pay attention that because the sedimentation depends not only on the molecular weight, but also on the shape of the particle, the sedimentation of the shape of the particle, the sedimentation of the small subunit 30 S and of large subunit 50 S does not add to 70 S, which is the sedimentation of the entire bacterial ribosome. So very often you will see these numbers because they're easier to remember than the exact molecular weight of these large particles, and these values were described before molecular details about the composition of ribosome was established. So ribosomes consist of, and that is very, very important, both RNA and proteins. So they have RNA components and protein components. 70 S ribosome in terms of molecular mass is two and a half mega Deltons. So that's what I was referring to. Many enzymes can be 25,000 Deltons in molecular weight, and here we're talking about two and a half mega Deltons. So orders of magnitude larger size can be seen in case of ribosomes. Entire ribosomes, two and a half mega Deltons, small subunit 930 kilo Deltons, large subunit 1.6 mega Deltons. The RNA component of the small subunit sediments at 16 S, 16 Svedberg, and this corresponds to 1,500 nucleotides. A large subunit has two RNA molecules, 23 S and 5 S. The larger one has almost 3,000 nucleotides, and the 5 S ribosomal RNA, it's referred to as ribosomal RNA, has 120 nucleotides. How many proteins are there? In the small subunit, 21. In the large subunit, 31 proteins. The only other number that you should remember, and that'll help you in understanding the relationship between the ribosomal RNA component of the ribosome and the protein component of the ribosome, is the relative ratio of protein mass versus RNA mass. And so RNA is present in two thirds of the ribosome. So two thirds of the ribosome corresponds to RNA in terms of mass. So here it's shown. 66% is RNA proportion of mass and 34% protein. So you see already now that ribosomes are mostly RNA based machines. However, a significant part of the ribosome is the protein component. So now that you have a sense about the shape and the size, the molecular mass, and the composition of ribosomes, I would like to introduce different key chemical features of protein synthesis process. And just to remind you, the process of protein synthesis starts with a messenger RNA molecule that is transcribed such that one of the DNA strands that is used as a template is transcribed. Once you have a messenger RNA sequence, translation will start at a particular codon. And this codon is also special because it means start translating. So the initiation signal is AUG. The amino acid, the ribosome will catalyze polymerization of amino acids into a growing polypeptide chain until a stop codon is reached and then protein synthesis will terminate. So how do these ribosomes read the messenger RNA message? So just to remind you, just the way we talked about the chemical directionality of the two strands of DNA, in terms of messenger RNA molecule, you can also talk about, of course, two chemically different bands, the 5' end and the 3' end. And so how do these ribosomes know in which direction to read, to translate this message? Well, there was a very simple experiment that was used to deduce the direction of translation, the chemical direction of translation. So what was found out is that if you use synthetic RNA molecules, very similar to those short synthetic RNA molecules that were used, that Marshall Nirenberg used to demonstrate that poly-U or triplet of U specifies a phenylalanine amino acid. Here, a slightly different messenger RNA was used. It was a poly-A and at the very end, at the 3' end, and this was chemically known, it had a C, this messenger RNA. So as a product of translation from this messenger RNA, people observed that they could get a polypeptide chain that had polylysine and an asparagine. So now, this experiment was, of course, done after all 64 codons were understood in terms of their meaning, so after the genetic code has been cracked. So what this meant is that AAA meant a lysine, that was clear, AAA meant lysine, but AAC, which would be the last codon, if the message would be read from 5' to 3' was an asparagine. And since the product was polylysine and then asparagine at the end of the synthesized protein, this meant that the ribosomes will read this message from 5' to 3', so in 5' to 3' direction. If the ribosomes would read the message from 3' to 5', then one of these amino acids would inform amino acids would in fact be a glutamine, because CAA codon would specify a glutamine. So very elegant, but not only did this experiment tell us that the ribosomes read the message from 5' to 3' direction, this experiment also told us that as the ribosomes start reading the message, the N-terminal amino acid will be the first one to be synthesized, because this protein product had asparagine incorporated, amino acid incorporated on the C-terminal end of the polypeptide chain, so biochemically this was shown. There was another very nice and elegant experiment that actually showed that the protein synthesis takes place from N-terminus to C-terminus and the way this experiment was done is that the cells were broken and they were exposed to radioactive amino acids and at certain time after the broken open cells that were still capable of translating proteins, after a certain duration of time, people would simply stop the reaction and analyze the the partially produced protein products. So what happened is that the, sorry, they did not analyze the partially produced products, but fully produced proteins, so proteins that were synthesized and released. So after adding radioactive amino acids to translating ribosomes that were reading the message and synthesizing proteins, after a few minutes, four minutes, they ended up with some of these proteins synthesized that were all starting at different stages, you know, some of these ribosomes were only beginning to synthesize protein based on the sequence in the messenger RNA and some were already towards the end. So those that were closest to the end, they released the proteins and the radioactive amino acids were then analyzed in terms of where they are in the polypeptide chain. Are they close to the N-terminal side or closer to the C-terminal side? And this is the analysis, so people would break open this polypeptide chain and simply separate the little peptides that were produced after cutting the protein. And those that were closer to the N-terminus were analyzed versus those in the middle versus those on the C-terminal side. And it turned out that after a short time, and this is the curve here, most of the radioactivity was found closer to the C-terminal end. And as the time went by, more and more of these completed chains had radioactivity, so a longer time passed. And then after 16 minutes, for example, there was still most of the radioactive amino acids on the C-terminal side, but more and more would also be found on the N-terminal side. That meant that you had this diagonal increase of the amounts of radioactivity spreading from the C-terminal side to the N-terminal side. And after 60 minutes, you see many of these fully synthesized proteins would have had fully radioactive amino acids. So this experiment showed us that protein synthesis happens in such that the N-terminal amino acids are synthesized first. So, what else is important in terms of the chemistry of protein synthesis? So, the next key concept that you have to understand about the synthesis of proteins is that the next amino acid that is added, so let's say this is the first amino acid and the next one that comes in that has to be added, is added to the N-terminal side. Such that a single amino acid is added in front of the growing polypeptide chain. So basically, the elongation of this chain happens such that a single amino acid is added, it's inserted between the tRNA and the previous amino acid. So, if you stop the protein synthesis in the middle, just the way you have seen in the previous experiment, then you will end up with, if you kind of split the process of protein synthesis in the middle, you will have tRNAs, each one of them with an amino acid brought next to each other on the ribosome, and then the two amino acids react. And the way they react is that the new amino acid is added in between the previous amino acid and the tRNA. So, you see here, the blue one is inserted here, the green one will be inserted here, and so the growing polypeptide chain starts with the N-terminal amino acid and successively additional amino acids are added and kept covalently attached to the tRNA molecule that is here. You will see this on multiple slides, so if you don't fully understand this, I think it will become more and more clear as we continue with the discussion of translation. So, let me introduce to you these stages of translation. We will talk about initiation, elongation, and termination. So, what is initiation of translation? So, if you have a long messenger RNA, it will not be read, and you will see many reasons why this is less efficient in terms of regulation, it will not be read from the beginning to the end. This message actually has much more information than just to specify the sequence of amino acids. So, this additional information in the messenger RNA, just the way transcription process involved finding the promoter site, during translation the ribosomes literally have two subunits, and then they have to find the right place to start translating the message. So, let's say if this is the beginning of the coding region of this message, this is where the ribosomes have to assemble, and then translation begins until a stop colon is encountered, then the ribosomal subunits come apart, and this is the role of the two subunits. So, they come together when they need to translate. So, a little bit you can think about it as a computer searching for a particular file on your hard disk. Basically, through its magnetic head, the hard disk will have to quickly scan the entire disk. It doesn't read every single file with every single byte in each file. It'll literally scan around and some signature sequence of bytes that indicates the beginning of the file will be identified, it'll look at a directory of where things are, it'll go there, and it'll start reading. So, the same works for the ribosomes. They will not start just anywhere, but they have to identify a place to start. And in bacteria, these start positions can be more than one on a single messenger RNA. This is referred to as polycystronic messenger RNAs.