Winter_2024_Bis2A_Facciotti_Slides_Reading_21
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So, what can we do with some of those temporary copies of a gene? Well, one of the things we can do is to read the information that's encoded in them and turn that code into proteins. This is that process of translation that we've been hinting about.
There's a key issue with translation. Transcription. Transcription and translation are similar sounding words. The process of transcription is a process by which you make a copy of a gene. Genetic code in DNA is encoded in a nucleotide language or with a nucleotide alphabet, A, G, C, and T. And you transcribe that to almost the same language. Nucleotides of RNAs, A, C, G, and U, right?
Both of those languages have nucleotide bases in their alphabets. There are four bases in both of those alphabets. And so, we're just basically like transcribing a piece of text from English and just making a copy into English, that you could then use. Alright, so you're getting grandma's recipe book. You're opening it up. You take that recipe book and you make a copy of a recipe that you can use while you're cooking, in the same language that the recipe book was written in.
The process of translation, on the other hand, is really converting between two different chemical languages. In one case, we have the nucleotide language of the genome, and of the RNA molecules. And what we wanna do is to take information that's encoded in the language of nucleotides and translate it into another language that's encoded by different chemicals, the amino acids, that make up proteins. It’s like taking the transcribed copy of grandma’s recipe and translating it into the meal.
So, we now have this problem where one code is encoded in the nucleotide language of RNA bases that has an alphabet of four letters. And we want to read that code and create something new out of it that’s expressed in a different alphabet, that of amino acids. And we know that there are 20 amino acids that we need to encode. So, we need some sort of dictionary that allows us to read, like multi-language dictionary that allows us to read the language that's written in nucleotides and translate it into something that's written in amino acids.
Now, there's a central problem to that act of translation. We know we've got in one case, we've got four letters that are used to write information. And the other case, we've got 20 letters that are used to write information. So, we can't have a strict one-to-one mapping between the languages. That is, an A base can't just map to an alanine amino acid. That G base can’t map to glycine. C mapping to cysteine. This one-to-one mapping doesn't work because we only got four bases and we can’t encode 20 different things with four letters in this way. So, you can't have a one-to-one mapping.
You can try a two-to-one mapping. You can imagine doing something like where you have two of these letters, A and G map to one amino acid. But you can't have that either because the two-to-one mapping only leads to 16 possible combinations of two nucleotides. That's not enough to encode all 20 amino acids.
It turns out that the smallest number of A, G, C, and U combinations that you can use to encode 20 amino acids is to have a three-to-one code, where three nucleotides map to one amino acid. So, three nucleotides mapping to one amino acids. If you've got four nucleotides and you can combine them in groups of three it turns out there are 64 different possible ways to do that. And that's plenty of combinations to encode for 20 amino acids. Nature's seized on that idea and it's invented something we call the codon, a group of three nucleotides that are used to encode a single amino acid.
Alright, so we've got our translation dictionary. That translation dictionary is going to be written in groups of three letters of nucleotides. And, those are going to represent, each one of the combinations of three will represent at least each one of those 20 amino acids.
Then we need a tool to convert one code to another. This is a physical process. It's not just like a little exercise that the cell does on a piece of paper. It actually needs to move a little molecular building blocks around and physically touch a piece of RNA to make a physical protein. It's not just exercise on paper. So, the cell needs a tool that is able to do the physical conversion of code; read this key or dictionary, and translate, do the translation between the language of nucleotides and that of amino acids.
That key, that thing that links those two languages is the transfer RNA or tRNA that we saw a little bit earlier. So, here's a tRNA. tRNA is, we look at the figure on the left here, is a single-stranded piece of RNA. Here we go, five-prime. We can trace it around 5-to-3, 5-to-3, 5-to-3, et cetera. And end up at the three-prime end. So, it's a single strand of RNA that folds up into this beautiful little structure here.
Functionally speaking, it's got really two important sites. One is down here called the anticodon, a vocabulary term I expect you guys to learn. On the three-prime end here it's got a site for the attachment of amino acids. What does that mean? It's got something related to the three-letter code in our nucleotide dictionary. And it's physically linked to an amino acid. In this way, this molecule is able to have nucleotide language on one end and amino acid language on the other. This anticodon is specifically linked to a specific amino acid. Alright, so this molecule does the pairing between the two languages. It is the translator molecule.
How do we get the right amino acid onto the right tRNA molecule? Well, that's a job for an enzyme called the aminoacyl tRNA synthetase. And I just show this to give you guys a reminder that a lot of the stories we learned earlier in metabolism are also applied here. In this case, the mechanism of this enzyme, the enzyme being depicted as this purple blob. So, it starts out here, empty. Its active site is quite complicated. And the way this enzyme works is that in the first step it loads up on energy, ATP, and an amino acid. In this case e.g. alanine that comes into the binding site. Those substrates come in and the enzyme catalyzes the hydrolysis of pyrophosphate, just like we saw earlier in DNA and RNA synthesis, and couples that energy release to the joining of the AMP to the alanine.
So we've got what we call them activated alanine. Why? Because we've got this energetic molecule AMP forming a new covalent linkage to the alanine. And basically, that link has stored some of that energy of the hydrolysis from the previous step to be used later. In a subsequent step. This charged tRNA synthetase binds a specific tRNA, tRNA where the anti-codon matches the alanine that's gonna be put on. It's an alanine specific tRNA enzyme recognizes that by having special amino acids here that are complimentary to this particular tRNA. That tRNA comes in. There's a group exchange here where the AMP comes off the goals and the alanine gets linked covalently onto the tRNA. So we have, we can chase, do this energy chasing story where ATP brings energy and initially comes into the binding site. Atp is hydrolyzed, pyrophosphate is released. That energy of hydrolysis is being used to drive the formation of a new bond between the amino acid and AMP. And then the next step, that energy is utilized by trading functional groups here the AMP leaves the alanine gets linked to the tRNA. And now we've got a charged tRNA that's ready to add an amino acid to the growing polypeptide. Alright. So, same stories, similar stories to how we’ve talked about ATP getting used. There's some hydrolysis, There's some energy storage in a new bond over here. That bond is getting traded for a different kind of bond. And finally, we get the product that we want. Alright, so that's the loading of the tRNAs.
And the cell has many tRNA molecules. It needs to encode at least enough of them to have one specific anticodon for each amino acid that needs to be encode. So, you have at least 20 genes in an organism that code different tRNAs, but they are often more that encode a variety of different tRNA molecules.
Alright, so how do we take that tRNA and use it to add new amino acids onto a growing polypeptide chain. Not remember the polypeptide. What is a chain of amino acids and N, C, C; N, C, C; N, C, C. Now we had like this, one by N, C, C, or two or three. And so that's what's being depicted here. Polypeptide with the carboxyl group on the end. Here's the carboxyl group. There's the rest of the polypeptide below. And so, what we want to do is add a new amino acid. Add a new amino acid to the end of this chain. So that's what's being depicted in this chemical reaction. Polypeptide with a carboxyl group. Here comes the tRNA molecule that is used to bring in the right amino acid. The tRNA that's been charged with an amino acid goes into a catalyst called the ribosome. There's a release of water and the polypeptide now has a new amino acid on the end plus a free tRNA that can go get recharged. This is the biochemical reaction that we will discuss when we are talking about growing the polypeptide.
This catalyst is called the ribosome, and it does all of this. I'm going to give you guys this figure here just to mostly for your notes, but note that it's a big complex. I'll circle it in blue. And it has what are called the large and small sub-units. These are shaded slightly differently. You can see them assembling here and kinda binding onto mRNA molecule that comes in one end and leaves the other. Remember this mRNA molecule carries the code for a protein. One thing I want to make sure you guys understand is that this ribosome is not turning this RNA molecule, this mRNA molecule into protein. The mRNA is just serving as a, as a repository of the information that's required to make the growing protein, which is what is depicted coming off the ribosome up here. Alright.
So, the ribosome is an amazing machine. Here's some structures of it from two sides. Note that it's pretty darn big and it's got all sorts of twisted, hairy looking stuff going on here. We’ve got a large subunit in blue and a small subunit and pink.
And if you look at it just right from one side, you can see that there's a channel in here into which the mRNA can fit. I do want to point out that the ribosome is a molecule that is made up of both protein and RNA.
We looked at the ribosomal RNA, at least a model of it earlier. But if I take this model here and I just show you the RNA component, that's what's over here on the left. All these are strands of RNA. Then packed in-between all the little gaps of this RNA are bunch of proteins here on the right, shown in purple. These kinda give stability and structure and other functions to this, this crazy looking machine.
Here, we put the protein and RNA together. We end up with the full ribosome with the RNA molecules in blue and magenta showing the protein molecules.
It turns out that one of the big discoveries that was made maybe 25 years ago or a little bit more than that is that the catalytic groups that are responsible for helping make new peptide bonds actually come, not from the proteins, but from the RNA component of the molecule. And that was a big surprise at the time.
If you want to hear more about the ribosome, I recommend watching this lecture that was given a couple of years ago over at the Genome Center by Joseph Puglisi. He has been spending years studying the ribosome and the process of translation. He gives a great talk that's really digestible. And it’ll give you a little bit more information, actually, a lot more information than we need for today, but it's really fascinating story. And you'll see a lot of familiar themes pop up as he tells the story of protein synthesis by the ribosome.
Alright, so we already talked about these a little bit before. But I want to remind you that just like in transcription, in translation we need start and stop signals. So, the transcriptional signals right, were the promoter to recruit the RNA polymerase and the terminator to stop transcription. These signals are encoded on DNA. In translation, the ribosome also needs some signals to start and stop. And these are encoded in the mRNA. AUG, the start codon tells the ribosome where to start adding new amino acids. And there are stop codons that are also encoded in the mRNA that tells the ribosome where to stop.
Again, I emphasize the point here that we want you guys to know which signals go with which process. The promoter goes with transcription. The terminator goes with transcription. The translation start codon goes with translation; the stop codon goes with translation.
In bacteria, there's an interesting story of how this whole machine comes together. It's a little bit different in eukaryotes, but I will show you guys the bacterial version here. Depicted here is a small subunit of the RNA polymerase. In comes the mRNA that's about to try and bind with the ribosomal subunit. And what's cool about what happens in bacterial systems, we've talked about this thing called the ribosome binding sites, a little sequence in the mRNA that is responsible for helping the ribosome bind the mRNA in the right place. It provides the initial alignment between the two molecules, mRNA and ribosomal subunit. And what you'll notice here is that the mechanism that's being used, a theme we've seen a few times before, is that we have homologous or complementary antiparallel base pairing. A, U, G. C, G with C, A with U, so on and so forth, between a specific sequence on the mRNA and a piece of the RNA of the small subunit of the ribosome.
Now, every now and then, I like to test students to see if they understand that this strand here is going five-prime to three-prime. What then do we expect the orientation to be here? (PAUSE) We expect it to be antiparallel. This functions by anti-parallel base pairing. So we expect this strand to go five-prime to three-prime, so that, that bottom strand is anti-parallel to the top strand that's coming in. That association provides an anchor and some alignment so that the start codon can align properly in the middle of the ribosome.
And, the initial tRNA that's loaded with an amino acid can come in. The tRNA can use its anticodon here and base pair with the codon on the mRNA through complimentary anti-parallel base pairing.
The rest of the ribosome can assemble around that. Then other amino acids can enter the ribosome. Get themselves aligned just right so that the previous amino acid is smuggled up against the incoming amino acid. And the chemistry - that we described earlier - can take place to join the growing polypeptide to a new amino acid.
Now, there's a lot of sites that describe this process and have you learned what the exit site and P site and A site and all that business before. I'm not going to be bothering you guys with that in this course. But those of you that go on and take BIS101, for instance, you'll start learning a bit more about the role of each of these sites in this process.
Alright, how do we stop this thing? Well, it turns out you can imagine this process happening. Amino acids come in, they're joined. This thing continues, chugs along the mRNA until it reaches the stop sign, the stop codon. It is induced to stop by the binding of something called a release factor. This is a molecule that comes in, can interact with the stop codon, and does not bring with it an amino acid.
It thereby stops the addition of new amino acids to the growing chain and terminates protein synthesis. The polypeptide is then released.
Now, one other thing I didn't mention yet. I want to just talk briefly about the energy source. Here’s that process depicted again. The ribosome binds a new amino acid. First amino acid, tRNA with the amino acid. Some new amino acids come in. There are these additional proteins called elongation factors. Ef, elongation factors, we denote them ts and t. But these elongation factors bring with them a source of energy. If you look at Jody Puglisi’s talk, he describes their action as basically coming in this process through the hydrolysis of ATP, giving the ribosome a kick to drive this process forward.
And then there's a little further down here another elongation factor that harvest some energy from GTP to kind of do the same thing at a different point in the process.
Alright, that's it for Lecture 22. The key things here we're to learn the signals of transcription and translation. Excuse my handwriting is deteriorating. Be able to make models of the transcriptional unit, both for protein coding and non-coding genes. We need to know which signals, by the way, which signals go with which processes. Often they're the confusing thing about this lesson. Know the rules. The different catalysts we talked about. The RNA polymerase, the ribosome, the spliceosome, the aminoacyl tRNA synthetase. What are these proteins doing and how are they working? Then just in general, be able to draw out the key steps in each of these processes. I think that would be the end. We'll be back on Friday to talk about gene regulation.