Fundamentals I

9-22-08 Part I

10:00-11:00

Dr. Miller

Slide 1: PROTEIN SYNTHESIS

Today we’re going to talk a little bit about protein synthesis. The value of that is it will present to you some rational for some of what I talked to you about before when I talked to you about energy conservation, energy buildup, energy utilization. When we synthesize proteins, we utilize a great deal of the energy that we have stored in ATP. As a matter of fact it takes 4 high-energy phosphate bonds to put one amino acid into a polypeptide chain. Just one amino acid going into a polypeptide chain takes 4 ATP equivalents. Actually it’s in the form of 2ATP’s and 2 GTP’s. But basically the situation here is the nucleotide-nucleic acid polymers generate amino acid polymers and that requires a translation. It’s like going from Chinese to English, German to Latin, or English to Latin or whatever you want. You have to translate the message of the DNA into the message of a protein.

  • The players in this ballgame are the messenger RNA’s, which are derived directly from DNA’s
  • tRNA’s which actually do the translating because they have a portion of them that’s capable of interacting specifically with a message. They also have the capability of carrying an amino acid to the right place dictated by the message.
  • We have aminoacyl tRNA synthetases. These are the enzymes that essentially look over the amino acid and they look over the tRNA, and they make the union of the proper amino acid with the proper transfer RNA. I can’t emphasize too much how valuable these enzymes are. There is only one for each amino acid, and that one synthetase enzyme has to choose the correct amino acid and the correct tRNA out of the hundreds of tRNA that are around. There are many tRNA’s that are utilized. Sometimes multiple tRNA’s are used for the same amino acid. So synthetase has to recognize first of all the amino acid and then choose out of a hundred or so tRNA’s the proper tRNA’s to which that amino acid should be attached.
  • Finally we have ribosomes, which are large particulate substances- particles. You can see them perfectly in the EM view of any cell. They are composed of ribonucleic acids (rRNA- ribonucleic acids) and protein. As a matter of fact the nucleic acid components are predominant. In other words there’s more nucleic acids in a ribosome particle than there is protein, which is kind of unusual. Over and above that this situation arises such that one of the RNA molecules here, which is in the ribosomes, is actually the catalytic agent for the synthesis of the peptide bond. When new amino acid comes into the protein to be attached to the growing protein, the enzymatic activity is performed by rRNA- not a protein. One of few examples when catalytic event is not controlled by protein.
  • So that’s kind of an introduction, and we’re going to look at all of those species in the course of the next hour and a half or so. We’ll take a look at what they do and how they do it.

Slide 2: Genetic codes as they occur in mRNA’s

  • First of all I want to remind you that the genetic code can be different in different organisms.
  • The code is the same but the way the code is used is somewhat different.
  • If we’re dealing with bacteria and some lower organisms, we would have an overlapping code where the first three nucleotides designate one amino acid, and, and the next amino acid is added to the protein by virtue of the 2nd, 3rd, and 4th, so that the 1st codon overlaps with the 2nd codonand 3rd and 4th all the way down the line…
  • You don’t have 3 separate nucleotides for each amino acid. That process is used, as you can imagine, in organisms such as viruses and some of the more primitive bacteria where there’s not a great deal of room for nucleic acids. You have to utilize nucleic acids very sparsely, very rigorously.
  • Our situation in mammals and our close relatives is such that we have nonoverlapping code. We have 3 nucleotides that specify one aminoacid. Then there’s 3 more nucleotides…4,5, and 6 for the 2nd amino acid. Seven, 8, and 9 for the 3rd amino acid and so forth. So it isnot an overlapping code.
  • Each amino acidis put in there by virtue of its own 3-letter code.
  • Codes can be continuous as they often are in some organisms, but they can be discontinuous as they are in our situation where we have messages that are synthesized. Initial messages are synthesizedand then spliced to provide mature message. We have exons, which are coding, and introns, which are noncoding areas. That’s an example of that. (Part b of the diagram where there is a dot specified as the comma.) Even though it’s one nucleotide here is the comma. In actual messages in mammals, the comma can be very, very large. It can be thousands of nucleotides.

Slide 3: The general structure of tRNA molecule.

  • Alright, the tRNA is often viewed in this type of picture which looks like a 4-leaf clover, if you will. It’s really a good way to look at tRNA.
  • The amino acid will be attached to the 3’ end- actually to the 3’ribose hydroxyl on that nucleotide. It’s always an adenine as base at 3’ end.
  • Then there will be a variety of locations around a tRNA, which by the way is only about between 70-80 nucleotides long. These are very small agents in so far as nucleic acids are concerned.
  • You add the amino acid here (at the 3’ end). You detect the proper locationon the message by virtue of the anticodon which is 3 nucleotides located at this particular region (@ 35 on the diagram). Just as far away from nucleic acid as possible.
  • Then around a tRNA are locations that are quite variable. There can be a large loop here. The dots here indicate where there can be a nucleotide. The same over here. The D loop over here can be rather large. The TC loop is the thymidylate pseudouridine cytosine loop. It’s a kind of tRNA that’s kind of a strange beast because it has some thymadine in it. Thymadine is supposed to be only in DNA, but tRNA does have some thymadine in it.
  • There are some regions along here. For instance, if you see a Y, that will always a pyrimidine. If you see an R, that’s the location where there will always be a purine. Phi is pseudouridine and all the way around.
  • You can look at tRNA and pick out these kinds of domains that are always the same, although they can be a big loop, a small loop. It can be a large loop here or a small loop.
  • Now, this, as I said is not really a good look at what the molecule really looks like. I will show you later the situation where the D loop has been wrapped around behind, and the TC loop has been looped over the top. That’s what the actual structure of the molecule is, and I’ll show you that a little bit later.

Slide 4: Identity Elements

  • First of all we need to look at the regions where a synthetase enzyme will look and some of the enzymes that tell the synthetase enzyme when it’s in the right domain are found, for instance, obviously in the anticodon, and here and here (where the other two orange dots are.)
  • In this case now we’re talking about the yeast enzyme, which is going to Phe on this tRNA.
  • The enzyme will look to make sure that is has the right tRNA. It will look at the anticodon, and it will look in the D loop. If that all is proper, it will accept that tRNA for the addition of Phe.
  • Now, if we’re talking about Met, to be incorporated into the protein, it’s an interesting situation that only the anticodon is the important thing there. The reason for that is that Met is a very special amino acid. It is always the very first amino acid that is placed in the growing polypeptide chain. I don’t care whether you are looking at the lowest organism or you are looking at yourselves, you will synthesize every protein that you make, every protein that is synthesized by ribosomal synthesis mechanisms on this planet begin with Met. Met is often removed in the functional protein because the signal peptide and other peptides are removed, propeptides. But, Met is the first one.
  • Methionine has unique codon and a unique anticodon, and that’s all the enzyme has to see is that Met codon, and BOOM- It will accept it as the right tRNA for Met.
  • Now for Ser the situation is a little more complicated. The anticodon isn’t that important anymore, but the stem regions up here- the stem of the tRNA is what’s important as well as 2 locationsin theD loop.
  • For Ala, the synthetase will look at the stem region, but not as widely as stem region for Ser. Likewise here there’s a big loop here and only a small loop here. (Referring to the loop regions of the Ser and Ala tRNA’s in the diagram.)
  • There are signals on these tRNAs which allow them to be taken into the province of a given synthetase.
  • Remember there’s only one synthetase that has to go andevaluate all the tRNAs that are around and take only those which are proper.

Slide 5: Ribbon diagram of tRNA tertiary structure

  • This is a view, a real view of a tRNA molecule. 3’ end here, 5’ end here, and it winds on down through.
  • Here are the anticodons, and some of the amino acids which are important which will be that the tRNA can carry, and are marked for by the anticodon are all of these amino acids here. (At the bottom where they are labeled anticodon.)
  • And some of you who remember from a few weeks ago, can anyone tell me which amino acid is designated by Y?

Answer: Tyrosine

  • Tyrosine! Alright, ok, Fantastic! That is absolutely marvelous.
  • How about D? Aspartic Acid Hey! Great! Man oh man, you can tell I was here. This is really wonderful.
  • So, you can also look at other areas. For instance this particular region here is important for these amino acids, this region for these amino acids, (The amino acids in the boxes.) And good old Phe here is important for this domain. (Shown in the Variable Loop in the Figure.)
  • So that is… I point this out because I want to give you the idea of how important those synthetase enzymes are. What a big job they have. They are really essentially the 2nd DNA, because if they make a mistake, the problem goes into the protein. If they make the wrong association of an amino acid with the tRNA, then that particular protein is going to be compromised because there will be a mistake in its sequence. It’s not as bad as a gene mutation because if you make a mistake here you involve only one example of the polypeptide chain where as you’d be making 10 million of the proper chains. There is a probability, which we’ll discuss, a little bit later, which mathematically you can evaluate how often a mistake will be made. It’s surprisingly rather large.

Slide 6: How Is an Amino Acid Matched with Its Proper tRNA?

  • The amino acid is matched with its proper tRNA.
  • Aminoacyl-tRNA synthetases interpret the second genetic code and they then discriminate between the various tRNAs and amino acids. That’s what I’ve been preaching to you for the past 5-7 minutes, so you know about this already.

Slide 7: The aminoacyl-tRNA synthetase reaction.

  • Here’s what essentially is done. The overall reaction goes essentially from the amino acid is identified and the tRNA is identified.
  • You use ATP, and you hydrolyze it not to ADP but to AMP. In other words the first event that happens to ATP in this whole process is the loss of pyrophosphate- 2 phosphate groups from the phosphate high-energy bond.So pyrophosphate is eliminated and use of Mg as a cation as a coenzyme or facilitator. The ultimate activity here is that the amino acid is esterified to the tRNA. This would be a carboxyl esterification to the hydroxyl on the ribose in the 3’ domain of tRNA. So that’s the overall reaction.
  • Now, how does this come about?
  • First of all the ATP hydrolyzed to pyrophosphate and AMP, and what is done then is that the amino acid is still attached to the enzyme and it is linked to AMP here (adenosine monophosphate) by a mixed anhydride bond. Carboxyl group and phosphoric acid being combined to make an anhydride group. That is a very high-energy bond. It has required to make it two hydrolyses: one involving pyrophosphate from ATP, and the other one which isn’t shown here is the hydrolysis of pyrophosphate to two inorganic phosphate molecules.
  • So you’ve actually driven this reaction. You’ve created this high energy bond here by hydrolyzing a one ATP molecule twice, which is conventional. That’s kosher. There’s no problem with that. This happens. But you have used 2 high energy phosphate bonds to get that process.
  • You’ve created a bond here that’s now going to containthe energy for all the subsequent reactions. It contains the energy to make this amino acid hook on to a tRNA by virtue of an ester bond. It has the energy also to make this amino acid to hook on to a growing polypeptide chain by a peptide bond. So you’re going from an anhydride, mixed anhydride to an ester to a peptide. That is going from high energy intermediate to low energy.
  • The sacrifice of one ATP here is quite worthwhile. It sets up the all the other reactions for the amino acid.
  • The amino acid is hooked to AMP, and what happens then is the tRNA grabs hold of the amino acid and takes it away from adenosine monophosphate by virtue of the oxygen. The free electrons on O here making a nucleophilic attack on the carbonyl group here & the amino acid winds up esterified to tRNA. This is the adenosine, and the amino acid is hooked on to tRNA by this particular ester bond here. We now have it that way.
  • There is another way to get an amino acid attached to tRNA. If 1st attack on the part of the tRNA molecule is from the 2’ hydroxyl here. In other words the 2’ hydroxyl esterifies the amino acid, and the tRNA is now holding the amino acid by an ester bond linked to the 2’ of ribose molecule. That is transferred however always to 3’ so that you wind up, no matter what class of synthetase is working, whether it attaches the amino acid first to the 2’ or at the 3’, ultimately everything ends up at 3’ location on the tRNA molecule. This is considered a charged tRNA. The tRNA (which is designated this whole thing, and this shows you only the very top part where the phosphate group is, and then the ribose is attached to the phosphate, and the amino acid is attached to the ribose.) This is called then a charge tRNA. The tRNA has been loaded with an amino acid, and it’s charged so to speak.

Slide 8: CODON – ANTICODON PAIRING

  • We now have the problem of hooking up charged tRNA to the various messages. That is a situation that is quite acute, and it’s something you should understand fairly well because it’s essential to the whole issue here.
  • We can charge tRNA with a lot of amino acids, but this doesn’t amount to a hill of beans if you can’t find the correct place where that tRNA is to carry that amino acid. That is done by recognition on the part of the tRNA with a message.
  • You know enough about nucleic acid chemistry to realize that you always read nucleic acids from 5’-3’ end…right? 5’-3’.
  • Also, when nucleic acids interact they interact in an antiparallel fashion.
  • So if your message is going from 5’-3’ in this direction, our anti-message or the tRNA carrying the anticodon will go from 5’-3’. That means that at position #3 in the codon interacts with pos #1 in anticodon. Position 2 in the codon with 2 in the anticodon, and position 1 in the codon with position 3 in the anticodon.They are running in opposite directions.
  • We always talk about nucleic acids running from 5’-3’.
  • Now I also have something here that is sort of an oxymoron in the sense that I’m calling this (5’) the aminoterminus and this (3’) the C terminus. Now the reason I did this is I’m not mixed up. I haven’t lost my mind. This is because at this part of message is where amino terminus of the polypeptide chain is made. A polypeptide is synthesized from its amino terminus to its C terminus, and by the time this message is read all the way at the end, you’re synthesizing the carboxy end of the molecule or of the protein. At the beginning you are synthesizing the amino terminal end of the molecule. As you gradually go down the line you go from the amino terminus, and eventually the C terminus is the last part of a polypeptide chain,which is put together at the C terminal end. This is true for all ribosomal synthesis no matter whether it’s done in a single celled bacterium or you. That’s essentially how that’s done.
  • The number of tRNA’s for amino acid is not the same as number of codons. There are more tRNA’s than there are codons.
  • Therefore the genetic code is degenerative- It’s not absolute. It’s absolute for Met and Trp, but not for the other amino acids. You can have more than one tRNA for the same amino acid.

Slide 9: WOBBLE POSITION RULES