This module is about the genetic code and specifically how the genetic code was actually broken by some very classic early molecular biology experiments. First of all people believed that genes and proteins were colinear, and what that meant was that if you have longer genes, genes that occupy more space along the double-strand, the double helix, that they would code for longer transcripts, and then when the transcripts were translated, they would code for longer protein. So here we see 3 genes of different length along the double-helical DNA molecule transcribed into 3 different length green mRNAs, and then translated into 3 different length polypeptides, that have now folded up. So in other words, longer genes encode bigger proteins. So if genes and proteins are indeed colinear, one is free to speculate as follows: Obviously one base couldn’t mean one amino acid. Otherwise, you would only be able to code for 4 possible amino acids. You wold only get 4 codons. Two bases – you’ll only get 4 squared, or 16
possible code words. And therefore you could only code for 16 amino acids. And of coursewe know that proteins are made up of 20 different
amino acids. Imagine that a code word was made of 3 bases. Then, you could get up to 64 possible amino acid codons. And that was enough to account for the 20 amino acids; that if you had 4 bases per amino acid, that would be 256 different possible codons, and that’s way more than enough to get 20 amino acids. And while it wasn’t formally excluded, it didn’t seem reasonable to go beyond a triplet codon. So now let’s talk about how the code was
broken. It was Marshall Nirenberg and his colleagues who broke the first few code words by identifying what fraction in an isolate from E. coli cells was actually the mRNA fraction. So they took E. coli cells and they burst them open; and they lysed them. They got a lysate and the first thing they did, which was very dramatic at the time, was to add 19 amino acids… and a 20th one that was radioactive, along with ATP on the grounds that making polypeptides would probably consume a great deal of chemical energy: and lo and behold, they produced radioactive polypeptides. This was called life in a test tube when it was first performed. It isn’t quite that dramatic, but it was the first demonstration of an ability to synthesized proteins, polypeptides in a test tube. You could then fractionate the E. coli cells into various components as you know, by cell fractionation. So the first thing with E. coli is to separate at high speed in an ultracentrifuge what was called the microsomal pellet, that is, the small bodies of the cell, from whatever was above in the supernatent, a clear cytoplasmic liquid called the cytosol. Then Nirenberg did several interesting things. To the microsomes, he applied an RNA extraction protocol which separated some RNA from the ribosomes, but without disrupting the ribosomes. And then he spun them again in the centrifuge at very high speeds and got another pellet. This pellet turned out to be ribosomes, but without some of the RNA. This RNA from ribosomes, shown here as fraction 2 was not ribosomal RNA. So the structure the ribosome was intact, but some kind of RNA had been taken off of it. The supernatant was also extracted. Specifically RNA was extracted from the cytosol, to get… well, let’s call it RNA from cytosol fraction 3, and then, an RNA-free cytosol. These are the 4 fractions that Nirenberg ended up with. He then did a reconstitution experiment, something we’ve seen before. He found that if he took the ribosomes and the rRNA that had been extracted from ribosomes, added them back together and threw in 20 amino acids and ATP (the same sort of experiment he did in the first instance) he did not get any polypeptide synthesis. In fact, in fact he tried various combinations as you see here, and got no polypeptide synthesis until in fact, he added back all four components. At that point, adding ATP is a source of energy (free energy) for
this process of protein synthesis, along with the 20 precursor monomer amino acids, he did get polypeptides. He did synthesize polypeptides, radioactive proteins basically. so it turned out you couldn’t leave out any fraction in this experiment. You may suspect as Nirenberg did, that fraction 2, this RNA that could be extracted from ribosomes, but that was not rRNA, might be mRNA. So Nirenberg did a very clever thing. He contracted with a fellow at Harvard to synthesize a ‘monotonous message’ called PolyU, which is just a string of uridines (uracil + sugar + phosphate), UMP, UMP, UMP, UMP, – a big string of uridine monophosphates, which is essentially a fake mRNA, right? And he added that instead of the RNA that he suspected might be real E. coli message. That’s fraction 2! So now he added back ribosomes (fraction 1), RNA from the cytosol (fraction 3) RNA-free cytosol (fraction 4), and poly(U)…, And one by one in what amounts to 20 separate experiments, he threw in one of the amino acids and ATP. A polypoeptide was indeed synthesized, but only when one amino acid was added, and that was phenylalanine. He may polyphenylalanine in this system, from which he concluded that the triplet code word UUU must code for phenylalanine. Nirenberg and his colleagues deciphered several other codons, the other poly-mononucleotides as well as poly dinucleotides and even poly trinucleotides, but that became pretty tedious after a while, and Marshal Nirenberg hit upon a method to decipher all 64 codons im record time. It turned out that if you took cells and you added radioactive amino acids to the cells, and you waited a very short time and then homogenized the cells so you end up with an homogenate, and then fractionated the cell into RNA from the cytosol, RNA- free cytosol and whatever was left in the cell, the rest the cell, it turned out that the radioactivity, which was radioactive amino acids, was not found where you would have expected it. Which is to say they weren’t in the RNA-free
cytosol. Instead most radioactivity was associated with an RNA fraction from the cytosol, the one we know as tRNA. Remember the RNA from the cytosol was an RNA that Nirenberg extracted previously from cytosol, so that he ended up with RNA from cytosol and RNA-free cytosol. Go back a few slides and you’ll see that. We know that now to be tRNA. In the old days by the way, that RNA was referred to as sRNA. Because it was from the soluble compartment, of the cell (the cytoplasm) it was called sRNA. Shortly after referring to this as sRNA in publications these RNAs were identified as tRNAs. So the amino acids that enter a cell rapidly associate with tRNAs. Before I go on show you how this enabled Nirenberg to break the genetic code in record time, let’s go through the aminoacyl transferase, or aminoacyl tRNA transferase catalytic reaction that associates tRNAs with their amino acids. It happens in two steps. The amino acid plus ATP (it’s going to provide energy
eventually) plus the appropriate aminoacyl tRNA synthase forms an aminoacyl AMP enzyme complex, liberating a pyrophosphate. So the free energy is actually captured now, as this aminoacyl AMP enzyme complex. Now a tRNA comes in, recognizes this energy-rich aaAMP enzyme complex, am finishes the reaction by binding the amino acid to itself, that is to produce an aminocyl-tRNA, liberating the AMP, disconnecting it in other words, from the enzyme, and of course regenerating or liberating the initial enzyme itself. So we can sum up those reactions as an amino acid + a tRNA + ATP becomes aminoacyl tRNA + AMP + pyrophosphate Polypeptide synthesis, which will be looking at, is one of the most expensive biochemical reactions cell can do. And one of the first expenditures of free energy is this ATP that has to be consumed to make every aatRNA that’s gonna be used in translation. Back to Nirenberg: He realized that ribosomes might be induced to associate with synthetic mRNAs as short as 3 bases. In other words he might be able to show ribosomes associating with, literally, a codon, a 3-base nucleotide, polynucleotide, a 3-base nucleic acid. And if that were possible, he might be able to show which codon enabled which aatRNA (aminoacyl tRNA) to attach to to a ribosome. So here’s the experiment: He found a filter through which tRNAs or aatRNAs, would simply pass in solution, also individual triplet codons would also pass through this filter. And so he had all 64 triplets synthesized, and then isolated an extract of E. coli which became all the aatRNAs (but he produced
all the triplets), and demonstrated that the triplets and the aatRNAs would indeed pass through the filter. He also demonstrated that if he isolated ribosomes (E. coli ribosomes), that they would not pass through the filter. They would be retained in the filter and they would stick on the filter. So the experiment then was to mix UUU and extracted ribosomes with the 20 different aatRNAs. This could actually be done in a single experiment in a single tube, unlike the original synthetic message experiment. You could mix these all together (think about why that was possible), mix them in the tube and then pour that stuff, after a moment or two, through the filter. The expectation (or the hope) was, as shown here, that the ribosome would in fact bind to UUU, and if it did, that the phenylalanyl-tRNA shown here, would be induced to bind to the ribosome via the UUU. via its codon. And if it did, then phenylalanine-tRNA bound to the ribosome could not pass through the filter and the filters could be analyzed to see if there was in fact such a complex. And indeed that was what was found. So UUU on the ribosomes would attract from such a mixture, only phenylalanyl tRNA. The remaining codons were quickly deciphered in the same way. Okay? So that’s how the genetic code was ultimately deciphered in short order. And here is the genetic code, all broken for you. So, this is one of many ways to represent a table of code words. But let’s look at some of the highlights here. first of all there are 3 code words that did not capture any aminoacyl tRNAs. And these turn out to be ‘stop’ codons.
These are 3-base protons that would be found near the end of a mRNA. It would be an indicator for the ribosome to disassociate and fall off, because the polypeptide it was making was finished. The stop codons are: UAA, UGA, and UAG. I’ll show you how you read this particular genetic code dictionary. Watch the order
of nucleotides or bases lighting up here in the next click. First there’s an A, then there’s a U and then to the right near the top there’s a G. That AUG, right? first base is A 2nd U, 3rd is G, AUG is the code word for methionine. And as you will learn shortly (or perhaps remember), methionine is the first amino acid put into all polypeptides, (on this planet anyway). So it is the start codon. I think I threw in another one here as an example: UAG codes for tryptophan. So if you needed a little more coaching on how to use this genetic code dictionary, you can look at that. UAG codes for tryptophan, nothing special about the tryptophan… just wanted to show you how you would read the genetic code dictionary for a 2nd amino acid. You can now do that yourself, for a few different amino acids. So for example you can look at cysteine and you will see that cysteine tRNAs are expected to have ananticodon for cysteine in the so-called ‘anticodonv loop’. And let’s see the other one down here, the other one is histidine and glycine are shown as examples. You can look at the codon and see that the anticodon is the complement to the codon. So you can do that on your own, using this genetic code dictionary or the one in your textbook which is laid out differently, but does the same thing. Finally, tRNA, is the decoding device. It’s because at one end, it has a site to attach an amino acid and usually has one at that 3′ end, and at the other end of this molecule, when it is folded over, is the anticodon in what’s called the anticodon loop. So on the left you see the traditional cloverleaf structure of a tRNA, and on the right you see what emerges after doing X-ray crystallography of tRNAs; it’s kind of an upside down L-shaped molecule. The 3′ end is shown here. it doesn’t say 3′ but because it says amino acid attachment site, that is the 3′ end. And again opposite is the anticodon loop. take a quick look to the left one more time: You see a number of ball bases: There’s a Greek letter psi, there’s a Z there’s an m2-G (a methyl guanosiine), there’s an I for inosine, or inosiine (alternate pronunciations!). These are unusual bases in the RNA sequence, and these were put there after the molecule was transcribed. We did talk about transcription of the different RNAs, including tRNAs. These unusual bases represent post-transcriptional processing, and here you have an example some quite specific ones. And that brings us to the end of this presentation.