1. The Genetic Code links nucleic acid and protein information
Features of the Genetic Code
1. Three nucleotides code an amino acid
2. The code is nonoverlapping
3. The code has no punctuation
4. The code is degenerate
Lets talk a little bit about the genetic code, it is of course the relationship between the nucleotide sequence and the protein sequence, when nucleotides become RNA and ultimately amino acid sequences. There are some features that we can point out about the code without going through the tedium of figuring how these things were discovered, this is a real hot item in the early 60’s, discovering the secret of life as it was led to several covers of Life magazine, you saw the DNA double helix and you saw watson and crick and especially francis crick becoming a part of the daily news as we worked out the relationship between genes and proteins which wasn’t obvious at first and it was especially cricks adaptor hypothesis, this idea that there must be some kind of adaptor molecule that can recognize a nucleotide sequence and adapt that code or translate it to an amino acid sequence. It turns out there is no easy way (no way at all) for a nucleic acid template to directly bond to amino acids and put them in sequence, they don’t interact with one another so there had to be some kind of adaptor molecule and Crick predicted the existance of tRNAs and somebody else when out and found them and then there was the process of actually unraveling the code itself which was done using synthetic nucleotides (RNAs) of known sequence, so you take UUU, poly U and you put it in a protein translation mix that would take any RNA and translate it and they put UU in and saw that it made (phe, phe, phe) and so they knew that the codon UUU codes for phenylalanine and they basically played that game until they had all 20 amino acids worked out, we are not covering all of that but a little background on a very dramatic period of biochemistry and microbiology. So out of all of that came this information.
First of all there are three nucleotides for every amino acid, that is, it’s a triplet code. Its not overlapping, that is, you read three at a time, the next three codes for a different amino acid. It turns out that some viruses that are real clever and they do have overlapping codes. Everything in biology has an exception. Some viruses actually get 2 for 1, get a twofer out of a gene because they start at different places, but that’s a total exception, but for all practical purposes there is no overlapping. There is no punctuation, that is one of the nucleotide bases is not used, it delineates that triplet from another, you could imagine that three of the nucleotides actually code for amino acids and they fourth one is a punctuation mark that separates off the actual triplets but it doesn’t have that. All four nucleotides participate in the information transfer and the code is degenerate. What that means is that more than one codon, more than one triplet codes for a single amino acid, so that there is more than one translation of a single amino acid that is shown in the next slide.

2. If we look at for example, serine
If we look at for example, serine. Serine can be coded for by U, C, and U, we code for serine, but you can also code for serine through U, C, and C. Or for that matter U, C, and anything will give you serine so there are at least 4 different codons for serine, and I believe there are actually a few others where the second nucleotide is varied, so that’s what we mean by degeneracy. Its not a literal one to one code, that one amino acid can be coded for by different codons and you see that all over the place.

3. The code is universal, that is bacteria uses the same genetic code as we do, plants, everywhere you look it is exactly the same. The table on the previous slide codes for the same amino acids in all living organisms as far as we know with one exception which is our mitochondria for some reason have adopted an alternative form of the code. It is minor deviations, they are not really terribly important for our core purposes other than to recognize that mitochondria do things a little differently like they turn a stop codon into a codon for tryptophan. They change the codon from arginine into a stop codon and so forth.

4. Transfer RNAs are adapter molecules that connect specific amino acids to specific codons in RNA
Transfer RNAs have a common design
1. There is at least one tRNA for each aa
2. Each tRNA is a single chain of nts
3. All tRNA molecules are L shaped
So the adaptor molecule that Crick predicted turned out to be tRNA and as we were talking about earlier they all have a common design. It is a clover leaf structure that is L shaped in three dimensional space. By necessity there needs to be at least one tRNA for each amino acid because each tRNA needs o load that amino aicd onto a specific anticodon to recognize the codon in the message. They turn out to be pretty regular in size right around nucleotides long and some have extra loop structures that make them a little bit bigger.

5. So here is a three dimensional model
So here is a three dimensional model. It folds up with a lot of intramolecular base pairing in such a way that on opposite ends of the tRNA molecule you have the CCA terminus that is responsible for binding the amino aicid and then on the opposite end you have the anticodon loop which is a series of three nucleotides that complimentarily base pair with the codon, the sequence in the message that specifies that amino acid.

6. 4. tRNAs contain many unusual bases derived by modification
4. tRNAs contain many unusual bases derived by modification. Methylation prevents formation of certain base pairs, favoring alternative interactions. Methylation increases hydrophobicity and interactions with proteins
We have already talked about the unusual bases in tRNAs. You see dihydrouridine for example with its hydrogen's, the reduced form of uracil. 5-methylcytidine where cytidine is methylated, inosine instead of guanine and an adenine you do aminate to get inosine. So you see these funny guys in bases, so we talked about before they basically change the structure, the tRNA certainly very specific reacted with.

7. 5. All tRNAs have a clover leaf design with about half the nts in base paired double helices and the other half represented as 5 specific functional motifs: a. 3’ CCA terminal region of the acceptor stem b. The TyC loop contains a ribothymine- psedouracil-cytosine sequence c. The “extra arm” containing a variable number of residues d. The DHU loop contains several dihydrouracil residues e. The anticodon loop - complementary bonds to mRNA codons
There is functional groups, we have already talked about the CCA terminal region, it binds to the amino acid, the T pseudouracil C loop has the pseudouracil group in it and it is involved in interaction with enzymes that are responsible for loading the amino acid on to it as are the extra arm and the Dihydrouracil loop these are all specific strucures that differ from one tRNA from another and finally the anticodon loop which is unique to each tRNA and it has a complimentary sequence to the codon for the amino acid that it is baring.

8. So it kind of looks like this, there is the place the amino acid goes on the CCA that was put on there. The anticodon loop is opposite to it and contains the compliment to the codon.

9. 6. The 5’ end of tRNAs are phosphorylated and is usually pG 7
6. The 5’ end of tRNAs are phosphorylated and is usually pG 7. Activated amino acids are attached to the adenosine residue at the end of the 3’ CCA component of the acceptor stem 8. The anticodon is present in a loop near the center of the sequence
A 5 prime end is usually guanine and the activated amino acids on CCA and kind of opposite to the amino acid we have the anticodon loop.

10. “Wobble” allows some transfer RNAs to recognize more than one codon
Yeast alanyl-tRNA has the anticodon IGC which
binds to three codons: GCU, GCC and GCA
The first two nucleotides are the same whereas the third varies – it “wobbles”
The first two bases of the codon bind precisely so that codons that differ in either of the first two bases require a different tRNA
Part of the degeneracy of the code arises from wobble in pairing of the third base of the codon with the first base of the anticodon
Some of the degeneracy of the code is accounted for by “wobble” for example if we go back and look at serine (on slide 2) We see that U and C pretty much determine serine. If you have U and C you can have anythign in the third base and you will still get serine coded for, that is what we mean by “wobble” The first two bases basically determine the amino acid codon. So for example alanyl-tRNA for yeast has various codons that all match with IGC. So basically it is a two letter code with just an occasional use of a third nucleotide to distinguish tricky situations.
So the point is by having degeneracy it protets against mutation. In other words tha tthrid nucleotide can change and you won’t have any consequences. He doesn’t think that is the reason it evolved though, it probably evolved because there are 20 amino acids that were selected for that are functional that is they make good proteins, 20 natural amino acids that could be used and if you had a two nucleotide code that would be 4 to the second power, that gives you sixteen possible doublets and sixteen isn’t 20 so probably the reason we have a triplet code that is degenerate is because you need a little more coding capacity that you can get with two nucleotides so you go to the next number which is 3 and there are whole bunch of extra codons left over and they simply just get used for different amino acids in a degenerate fashion. So it is probably more like that instead of selective pressure.

11. Ok so the anticodon of the tRNA needs to make good antiparallel complimentary base pairs to the codon in the messenger RNA so you read the codons 5 prime to 3 prime and the anticodons are read opposite of that (3 prime to 5 prime) so they can be lined up in base pairs (AU and GC).

12. Amino acids are activated by adenylation
Amino acids used for protein synthesis must be
attached to specific tRNA molecules; this is the “translation” step of protein synthesis
The amino acid attached to a specific tRNA will then be incorporated into the polypeptide according to the anticodon on that tRNA
Energy must be provided to make peptide bond formation thermodynamically favorable
This is accomplished by the formation of aminoacyl-tRNA
Were coming probably to the most important part of protein synthesis, about the whole idea of having a genetic code in the first place and that is how the code is decoded. We often think of the ribosome as being the structure in the cell that decodes the code, it takes messenger RNA and turns it into protein and the assumption is that somehow the ribosome did the decoding but that is a falacy and if you learn that, get that out of your head because all that ribosomes do is take any ol’ RNA and in a very systematic way match that Rna to tRNAs that have amino acids on them and whatever is on that tRNA, that is what you get in the protein and in fact ribosomes can be made to incorporate amino acids that are not coded for by a tRNA. All you do is take the tRNA, change the amino acid that is on it to another amino acid, you can do that chemically, and if you put that into a ribosomal mix it will put that amino acid in, it didn’t decode anything. So the question comes up, how did you decode from a nucleic acid sequence to a amino acid sequence, who is the decoder? And that turns out to be these enzymes that match the correct amino acid to the correct transfer RNA. This is the actual translation step when an enzyme grabs onto alanine and grabs on to alanyl-tRNA and charges, that is bonds that alanine to the 3 prime TCA group the translation has occurred. Everytime one of those tRNA synthetases does that it is decoding and then the charged tRNA comes to the ribosome and puts whatever is on there in. So these amino tRNA synthetases become the focus of our attention. They need to do a couple of things. They need to charge the amino acid but they also need to bring in enough energy to drive the synthesis of a peptide bond so they need to be high energy molecules (precursors) so that’s what we have in the form of aminoacyl-tRNA what we mean by that is the tRNA with an amino group on it and an acyl linkage.

13. Aminoacyl-tRNA synthetases attach amino acids to the 2’ or 3’ hydroxyl group of the ribose unit at the 3’ end of tRNA
So this happens by attachment to the CCA groups on the end of the tRNAs by binding the amino group to one of the hydroxyl groups of the ribose units so the 3 prime end.

14. The first step of the aminoacyl-tRNA synthetase reaction is the formation of aminoacyl-adenylate from an amino acid and ATP amino acid + ATP  aminoacyl-AMP + PPi
This is the aminoacyl-adenylate precursor that is used to synthesize aminoacyl tRNAs in the enxt step so in order to provide the energy to activate the amino acid so It can be added to the tRNA you need to create an aminoacyl-AMP intermediate or an aminoacyl-adenylate. So ATP is reacted with the amino acid, pyrophosphate is cleaved off, so that guy is going to have to be cleaved to inorganic phosphate in order to drive the reaction forward and we now charge the amino acid by bonding it to AMP, we have a high energy phosphate bond there that can be used to drive the synthesis of acyl-tRNA. So this is the first step of charging tRNAs is to activate the amino acid.

15. The second step is to transfer the aminoacyl group of aminoacyl-AMP to a specific tRNA to form aminoacyl-tRNA aminoacyl-AMP + tRNA  aminoacyl tRNA + AMP DG0’ of this reaction is close to 0, therefore pyrophosphate must be hydrolyzed to drive the reaction Thus the overall reaction is: amino acid + ATP + tRNA  aminoacyl-tRNA + AMP + 2Pi
Then the second step is to transfer that to the tRNA. This is the enzyme aminoacyl tRNA synthetase. The aminoacyl-AMP reacts with tRNA, the synthetase transfers the amino group to the tRNA and releases AMP. Delta-G for the reaction is pretty much 0, so it is the cleavage of the pyrophosphate in the previous step to pull this reaction forward and make sure this happens.

16. Aminoacyl-tRNA synthetases have highly discriminating amino acid activation sites
Because aminoacyl-tRNA synthetases perform
the actual translation process they must be
be highly specific for their appropriate amino acid and cognate tRNA molecule
Thus there must be a different aminoacyl-tRNA synthetase for each different tRNA molecule
Different aminoacyl-tRNA synthetases recognize
specific tRNAs via interactions with their anticodon loops and acceptor stems, especially modified bases and ribose units
So that’s the enzymes here, aminoacyl-tRNA synthetases and it follows that since they are charged with specifically bonding a given amino acid to a given tRNA there needs to be at least 20 of them. There needs to be at least one synthetase for every amino acid and there are actually redundancy there, some amino acids have more than one synthetase and they are going to do the translating process. They grab a hold of a specific amino acid and bind it to a specific tRNA, they can do that because on their surfaces they have binding sites for alanine for example and then they have a binding site for alanyl-tRNA with all of its idiocincricies and it reacts only with those two molecules and then catalyzes with the formation using the amino-adenylate form of the amino acid, the formation of the aminoacyl-tRNA. So this is where those acceptor modifications and acceptor stems become important.

17. If we look at consensus sequences for a bunch of tRNAs, we see these yellow regions that indicate hot spots, that is where most of the differences between tRNA molecules occur and therefore these are the regions that are being specifically recognized by the aminoacyl-tRNA synthetases. They tend to focus on the anticodon loop which you might expect. That is where the actual decoding process is occurring, but other parts of the molecule as well.

18. Ribosomes are ribonucleoprotein particles composed of two subunits
Ribosomes constitute about 25% of the mass of an E. coli cell
Ribosomes dissociate into a large subunit (50S) and a small subunit (30S)
The large subunit contains 34 different proteins plus two RNA molecules (23S and a 5S)
Lets start our discussion with the mechanism of protein synthesis. We have seen how tRNAs can be charged appropriately and how in fact the genetic code is decoded by these amino tRNA synthetases and now we are going to look at the mechanics of actually assembling these probably charged tRNAs into an assembled protein and of course this happens with a ribosome. Ribosomes are especially abundant in cells involved in protein synthesis in some cases the ratio will be even higher then 25% of the mass of an ecoli but even there that is pretty impressive of the ecoli cell a quarter of it is dedicated to the synthesis of proteins. Ribosomes dissociate into subunits in both prokaryotes and eukaryotes and there is a large one and a small one and in eukaryotes are the 50S and the 30S when they are dissociated and these subunits themselves are complexes (ribuonucleoprotein complexes) that contain RNAs as well as a certain number of proteins, generally proteins in eukaryotes then in prokaryotes making them bigger.

19. So here is the way it works is these individual subunits which actually exists ok so the ribosome is a dynamic structure in the cell and many of the ribosomes in the cytoplasm will acutally be present and dissociated into the 50S and the 30S subunits and only when they are presented messenger RNA and under the right conditions of assembly in other words they are initiation factors that make this happen will the active 70S ribosome form. When the two subunits come together on a message and then translation is possible.

20. Ribosomal RNAs are central to ribosome function
rRNAs fold into complex structures with many short duplex regions
Key catalytic sites in ribosomes are composed largely of rRNA; contributions from proteins are minor
So at the height of these machines is ribosomal RNA, for many years it was simply thought that rRNAs were a scaffolding (kind of frame work) where the proteins are deposited and the proteins that all the enzymology of loading of the amino acids and forming the peptide bond and translocating and all of that. We now know better, we know that the ribosomal RNAs themselves are clusters. They cluster certain sequences at catalytic sites. Probably in a similar way as we have seen with snurps, they use those sequences to interact in specific ways with their various nucleic acid substrates whether its mRNA or tRNA. The problem with ribosomes is that they are inherently complex and finding out the details of those functions turned out to not be simple but we are getting there.

21. This gives you some indication of the potential of rRNA to contribute to the structure. Not only is it a long linear sequence, it’s a specific nucleotide sequence and smart enzymes can bind to the sequence themselves but because of the formation of intramoleucular duplexes you end up with a lot of kind of like we see in tRNA, loop structures and large regions of double stranded structure and of course this all folds up and like tRNA folds up into an L structure it folds up into rRNAs, 3-dimensional specificity, they have very unique structures that can kind of act like proteins and we see a fair amount of base modification in the rRNA as well.

22. Messenger RNA is translated in the 5’ to 3’ direction
The direction of translation is 5’  3’
Thus mRNA can be translated as it is transcribed
In prokaryotes transcription and translation are
closely coupled in space and time
So we are going to talk about how these ribosomes function in a minute here but for now lets just get the basics of translation out on the table. mRNA is translated in the same direction it is synthesized 5 prime to 3 prime. This is one of the reasons why in a prokaryotic cell you can start translating a message before it is even been transcribed so a message gets halfway transcribed and this is happening in the cytoplasm of prokaryotic cell and the ribosomes immediately lock on and start translating protein before the message even gets fully produced. Everything is workin 5 prime to 3 prime in sequence, like train cars going down a track.

23. In fact if you take some ecoli and if you take a little dish detergent, some very clever scientist were able to very carefully splice the cell and place them out on a electromicroscope grid which shows the DNA being transcribed into these short RNA molecules. The RNA molecules get longer and longer as the RNA polymerase moves along the DNA template and as soon as you see any significant RNA out here it is clumped on by ribosomes so in fact it is not only possible but that’s what happen in prokaryotes, as soon as RNA is made it is being turned into protein.