DNA AND Genetic Engineering |Biomedical Engineering

Restored from the Empower Network archive (2011–2017), lightly edited to meet our current advertising standards. Views are the original author’s.

Professor Saltzman introduces the elements of molecular structure of DNA such as backbone, base composition, base pairing, and directionality of nucleic acids. He describes the processes of DNA synthesis, transcription, RNA splicing, translation, and post-translational processing required to make a protein such as insulin from its genetic code (DNA). Professor Saltzman describes the genetic code. RNA interference is also discussed as a way to control gene expression, which can be applied as a new way to treat diseases.
00:00 – Chapter 1. Introduction
01:35 – Chapter 2. Building Blocks of DNA
11:17 – Chapter 3. Structure of DNA and RNA
24:16 – Chapter 4. Central Dogma and DNA Synthesis
35:15 – Chapter 5. Genetic Code and Protein Synthesis
41:55 – Chapter 6. Control of Gene Expression

0:12 Professor Mark Saltzman: This week we’re going to0:15talk about DNA technology and genetic engineering:0:18this is Chapter 3 of the book. Some of this will be familiar0:22to some of you who’ve have had biology in high school or other0:26places, you know something about DNA.0:28In fact, even if you haven’t had a biology class it’s hard to0:32be alive in 2008 and not know something about DNA;0:36it’s become such an important part of our lives.0:41I’m going to ask you to indulge me while I go back to the0:44beginning and talk about some things that you know but I’m0:48going to go through this pretty rapidly.0:51I think the book has a fairly good description of it so if you0:54don’t pick up everything in the lecture,0:56hopefully you’ve read that beforehand, and you can go back0:59to it afterwards and read about things that didn’t make sense.1:03We’ll talk about some chemistry today,1:04what DNA molecules are like, why they have the behavior that1:07they do, and you need to understand this1:09in order to understand how you manipulate DNA.1:12So my goal today is to talk about sort of the basics of the1:16molecules, their chemistry, the function of DNA in cells,1:20sort of basic – the basic side of that.1:24Then on Thursday we’re going to start talking about how to1:29manipulate DNA and get closer to using it in Biomedical1:33Engineering. DNA is a double helix,1:36you know this, the double helix was–the1:39structure of DNA was discovered about the time that I was born1:42and so it’s been known throughout your lifetime,1:46you’ve always lived with it. It’s really remarkable how1:49far–how fast we have come from just knowing the structure of1:53this molecule to be able to manipulate it and study it in1:56great detail. I want to start by showing you1:59this cartoon that you already know about with the structure of2:02a double helix. It’s a twisted ladder and2:04there’s a couple of things to notice about this familiar2:08structure. One is that there are two2:10backbones right here in the light blue, so this would be the2:15upright parts of the ladder that are twisted.2:18Those are two continuous strands that wind around each2:21other to form the double helix. One thing to notice is this2:25part of the double helix that we’ll call the backbone.2:28The backbone’s on the outside of the molecule like the upright2:32struts of a ladder on the outside of a ladder.2:35On the inside are the rungs or the struts that hold the ladder2:39together. There are several things2:42that you’ll notice about the struts in this particular2:44cartoon. One is that there’s four2:46different colors and so you can see red, blue,2:49yellow, green here – four different2:51colors and that’s all there are, there aren’t more than four.2:55That there are two colors per strut, so what’s linking the two2:58backbones together are two colored segments that come from3:01the outside towards the middle, and that the colors occur only3:05in certain combinations, red and green,3:07yellow and blue, that’s all you see.3:10You don’t see a red and yellow, you don’t see green and blue.3:13This is a feature of DNA shown in this cartoon form,3:19so if you can keep that sort of schematic in mind,3:24it makes it a lot easier to understand the detailed3:27structure. That’s what I want to do for3:29the first few minutes of the lecture here is tell you a3:32little bit about the details of the structure and how molecules3:36fit into this image of DNA that’s already very familiar to3:39you. The things that you need to3:40know are the things that are really listed on this slide.3:43You’re going to know more details about it,3:46they’re not really colors, they’re chemicals,3:49specific chemicals – but the pattern is the same.3:53The molecules that really make up DNA are nucleotides and3:59DNA is a polymer of nucleotides. A polymer is just a large4:03molecule that’s made up of repeated units.4:05We’re familiar with polymers, plastics in our daily life;4:10the chairs that you’re sitting on are made of a kind of a4:15plastic polymer that is basically an organic chemical4:19that is cross-linked together. Cross-linked is not the right4:24word–that is chemically bonded with repeat units to make large4:28molecules so that when you have a bunch of large molecules4:32together they have certain physical properties like the4:36solid property of the plastic that you’re sitting on.4:40Nucleic acids, of which DNA is an example,4:44are polymers of nucleotides. So the repeating unit in DNA is4:49this structure here, a nucleotide,4:53which has three different regions.4:56There’s a sugar, a five carbon sugar,4:59which forms the core of the nucleotide and attached to this5:04five carbon sugar at specific positions on the sugar relative5:09to this oxygen, which is part of the sugar5:14ring, is a phosphate group and an organic base.5:20When these nucleotides get polymerized to form a long DNA5:23molecule they all get polymerized in exactly the same5:26way, the chemistry is the same. The phosphate group of one5:30nucleotide gets linked to the sugar group of another5:33nucleotide and I’m going to show you that in a few minutes.5:38So what’s going to form the backbone is this continual link,5:42phosphate to sugar, phosphate to sugar,5:45phosphate to sugar, all linked together to form one5:48long, long molecule.5:51What’s hanging off of the side of this long molecule that’s5:56formed by polymerizing nucleotides are – is this base6:00unit. It’s the phosphate and the6:03pentose that make up the backbone – that make up the6:07upright struts of the ladder and it’s the bases that make up the6:12connecting struts, so the bases are the colors.6:17There are four different bases, which I’ll talk about in a6:21moment. We’re going to talk about two6:24different nucleic acid molecules, two different nucleic6:27acid polymers, one is DNA, deoxyribonucleic6:30acid, the other is RNA, ribonucleic acid and one of the6:34differences between the two is that the pentose,6:38or the sugar, that makes up DNA is6:40deoxyribose shown here, and the pentose that makes up6:45RNA is ribose, shown here so every pentose in6:50a DNA polymer is deoxyribose. The phosphate is linked to this6:57carbon on the pentose, and notice that there is a7:00number on this carbon, it’s called – it’s the 5’7:04carbon. This is a convention that7:06organic chemist’s use when they’re describing molecules7:09like this. They’d like to be able to refer7:11to each carbon separately so they can talk about reactions7:15with this molecule, so they number the carbons.7:17In this case, these pentose molecules,7:20whether it’s ribose or deoxyribose, the carbons are7:24numbered the same 1′, 2′, 3′, 4′, 5′,7:27those are the five carbons that make up the pentose.7:32So I could refer to the 4′ carbon and you’d know I’d mean7:35this one, or the 2′ carbon you’d know I mean this one.7:38The ones that are important to us are the 3′ carbon and the 5’7:41carbon. The reason for that is that the7:445′ carbon is where the phosphate is attached.7:47In nucleotide the phosphate is always attached to the 5’7:52carbon. The reason that 3′ is important7:55is that when you polymerize two nucleotides together and a third8:00nucleotide, and a fourth nucleotide,8:03when you polymerize nucleotides together they get polymerized,8:06the phosphate of one gets linked to the 3′ carbon of8:11another. This is important because this8:14molecule here, deoxyribose,8:16is not the same upside down as it is – it’s not symmetrical8:21upside down and right side up, it’s different because the 5’8:25carbon’s either pointed up or pointed down.8:28The nucleotide has a directionality,8:31there’s an up and a down to it and it’s going to turn out the8:35chain that’s formed by polymerizing these has a8:39directionality as well and that’s important in defining the8:43structure. These are the pentoses -8:46remember 5′ and 3′ because that orients you with respect to what8:52direction the molecule is facing.8:55Now the bases, and you don’t need to memorize8:58the structures of these I’m going – I’m describing the whole9:01molecule to you in its molecular detail and then we’re going to9:05simplify it down to a version that we can talk about more9:08easily. To give you all the detail,9:11there’s two classes of bases that appear here.9:15One class is called the purines and they have two ring-like9:19structures. There are two of them that are9:22going to be important to us, one is adenine and the other is9:25guanine, shown here. Because saying adenine takes a9:29long time and saying guanine takes a long time we’re going to9:33simplify it by calling adenine (A) and guanine (G).9:36The second class is the pyrimidines and there’s three of9:40those that are important; uracil, thymine and cytosine9:43which we’re going too simplify by calling (U),9:47(T) and (C). Now remember that there9:50were only four different colors in the cartoon of the DNA double9:54helix that we talked about and I told you that those colors are9:59really – represent the bases but there’s five of them here.10:04There’s five because there’s one of these that’s particular10:07to RNA only, that appears in only RNA, and there’s one of10:10them that appears in only DNA. The one that appears only in10:14RNA is (U), the one that appears only in DNA is (T).10:18In DNA there’s only four colors, there’s only four bases,10:22(C)(T)(G)(A). In RNA there’s four bases10:27(A)(G)(U)(C). So (U) and (T) are10:31interchangeable in a sense that (U) appears where (T) would10:38appear in RNA and (T) appears where (U) would appear in DNA.10:45If I drew this altogether and this is one particular10:49nucleic acid, now shown in more detail,10:51all of the carbons of the pentose are shown here,10:55the phosphate is shown, and a base is shown.10:59This particular base is (A), this is ribose.11:05So this is a monomer or a single unit from an RNA11:07molecule, you know that because it’s ribose and it’s the11:11particular molecule that has (A) as its organic base.11:1411:18We don’t need to really talk about all the molecular detail11:22in order to completely describe a DNA or an RNA molecule because11:26these structures repeat themselves.11:29Every unit in the backbone of this ladder has the same sugar11:34unit, the same pentose; it’s either RNA or DNA and has11:38ribose or deoxyribose, so you don’t need to describe11:42the whole thing. You can just say it’s RNA or11:44DNA and you know everything about the pentose in every11:47molecule on the chain. You don’t need to say anything11:50more about the phosphate because they all have the phosphate and11:54every set of these is hooked together in the same way.11:58The 5′ carbon has a phosphate off of it and that phosphate is12:02linked to the 3′ carbon of the next one and they all have a12:06base hanging off the side. The only thing I need to say in12:10order to distinguish this particular part of the chemistry12:13of a DNA or an RNA molecule is to say ‘it’s DNA or its RNA’,12:16and ‘what the base is’. If I told you that ‘draw me a12:20nucleotide from RNA that has (A)’, you could go back to this12:24picture and you could draw the whole thing.12:28You can just talk about it in a simpler way.12:30You can say a polymer of DNA, for example,12:34is four bases long, that means it has four of these12:40repeat units and they go in the sequence from 5′ to 3′ of12:45(A)(G)(T)(G). If I told you that a DNA12:50sequence went 5′ to 3′ (A)(G)(T)(G) you could draw the12:54whole thing referring back to these notes, right?12:59In fact, you don’t have to draw it this complicated way,13:02you could just draw it as a line with (A)(G)(T) and (G)13:06hanging off of it, and you would know that that’s13:10a DNA molecule four bases long. Now what does the line13:14represent here? This represents the upright13:16struts on a ladder that I showed you before, it represents this13:19backbone that’s shown by – that’s formed by polymerizing13:23the pentose’s together through phosphate’s always going 5′ to13:263′, 5′ to 3′.13:28I could take and draw a line continually down the molecule13:31where my finger was touching; my finger would be touching a13:35phosphate here, the 5′ carbon,13:38the 4′ carbon, the 3′ carbon,13:40and the next phosphate. The backbone is what?13:45It’s phosphate, carbon, carbon,13:47carbon, phosphate, carbon, carbon,13:49carbon, phosphate and it has this structure hanging off the13:53side. Now DNA is a double helix13:56and I’ve only shown you one part of the helix,13:59right? I’ve shown you one upright14:01strut and a base hanging off of it, but it forms a double helix14:04because complementary strands of DNA strongly associate with one14:08another and that’s a very stable structure.14:11They do that because the bases can interact with one another in14:15particular ways, and this you know about.14:18This was the famous finding of Watson and Crick in describing14:22the structure of DNA. This is – what this diagram14:26shows you is–the forces that hold these individual strands of14:32DNA into a double stranded form. The forces occur because of14:37hydrogen bonding between complementary pairs of the bases14:42and the complementary pairs are adenine and thymine,14:46(A) and (T) and guanine and cytosine, (G) and (C).14:51Now if you read in the book, you read about where this14:53figure is shown in the book, you can understand more about14:56why these structures line up in the right way so that the right15:00molecular elements are together to form hydrogen bonding pairs15:05between them. That’s really beyond what I’ll15:08be asking you understand for the course but you can understand15:12that if you read it, I’m sure.15:14Now remember that (T) only appears in DNA and (U) appears15:20in RNA, and so (U) can also form a hydrogen binding pair with15:26(A). The whole structure of a DNA15:28molecule looks like this, going back to a more cartoon15:31version like I showed you before but adding some detail onto it15:34now. There are two upright struts of15:36the ladder, one shown in blue here, the other shown black.15:40They are linked together by four different colored segments15:44indicated here not by colors now but by letters.15:48It’s DNA, so it’s (G)(C)(A)(T) and they always occur in pairs.15:54Where before it was two colored pairs now it’s two lettered15:59pairs (G)(C)(T)(A). The chains are different16:04now. They were colored the same,16:06the backbones were colored the same in the diagram.16:08They’re colored different here to indicate one new difference16:11that you know about now, and that’s that there is an16:14orientation, there’s an up and down on the chain.16:17That’s due to the asymmetry of the nucleotide,16:20that there’s a 5′ and a 3′ end and the way that they’re linked16:24together. This blue chain here goes from16:275′ carbon all along the chain and there’s a 3′ carbon left16:30open at the bottom.16:3216:35If I wanted to link another nucleotide to this DNA chain16:38what would I attach here on the bottom?16:41I would attach the phosphate that’s connected to the 5’16:46carbon of another nucleotide. I would link this one facing in16:53this direction I would add onto this.16:58The other chain is facing in the other direction,17:00the 3′ carbon is up, the 5′ carbon is down.17:0217:06Remember that this molecule, let’s look at the blue one17:10wouldn’t be the same if I turned it upside down.17:15It wouldn’t be the same if I turned it upside down because17:18the carbons – the rings here, the pentose’s would all be17:22turned over, the chemistry would look different and the sequence17:26of bases would look different.17:2817:31The corresponding half of the ladder that corresponds to17:37any given ladder, let’s say the black DNA17:41molecule that corresponds to the blue one is not just a mirror17:46image. We call it the complement and17:50each strand of DNA, each polymer of DNA that you17:54could make or you could draw has only one complement and that18:00complement has the following features.18:03One, its chain is oriented in the opposite direction:18:08where this one goes 5′ to 3′, this one goes 3′ to 5′.18:13It’s oriented in the oppose direction and it has the18:17complementary base pairs at each position.18:20Where there’s an (A) here there’s got to be a (T) here,18:23where there’s a (T) here there’s got to be an (A) here,18:26where there’s a (C) here a (G), a (G), a (C).18:29That’s because you have to satisfy this base pair matching18:32in order to have hydrogen bonding in each of the struts of18:35the ladder in order to form a stable structure.18:3818:41If I’m talking about two DNA strands and they differ only18:45in one or two base pairs they won’t be exact complements and18:48they won’t form this double helix.18:5018:54This notion of complementary strands is very important.18:59It’s the way that DNA exists inside the cells of your body.19:04It exists in a double stranded form where every strand is19:06matched by its complement. These molecules of DNA,19:10very long molecules of DNA, are condensed and packaged19:15within the nucleus of every cell in your body.19:20Every cell in your body has exactly the same DNA;19:24that is if I could stretch out all the DNA and look at the base19:27pair sequence, the sequences of bases along19:31all the DNA in your chromosomes, they’d be identical in all the19:36cells. They’d be different in each of19:39us and that leads to the difference in the diversity19:42between people. You’ve heard about the human19:45genome project, we’ll talk about that a little19:48bit later. The goal of that was to take19:50for a typical human, or for a typical – in the case19:54of the human genome project maybe you’re looking at fruit19:57flies, you want to look at all the DNA20:00in a fruit fly, but to look at the sequence of20:03base pairs that makes up human DNA and write them all out;20:07we’ll talk about that later. This slide shows one20:10important feature of the physical chemistry of DNA that20:13turns out to be very important for all of the technology that20:17is built on DNA. It has to do with the nature of20:21this complementary binding between double stranded DNA and20:27the fidelity of this base pair matching in forming stable DNA20:33molecules. I told you that the fidelity is20:37very high. What does that mean?20:38That only strands that have this exact complement can form20:42double stranded DNA. Because of that you can do20:46the following experiment, and it’s a simple experiment,20:51it’s simple to understand, but the concept is very20:53important so I encourage you to think about it and make sure you20:56understand it. If I took two double stranded20:59DNA molecules and I exposed them to certain conditions that21:04caused them to denature, that means its native structure21:10falls apart. The native structure is this21:13double stranded structure here and if I heat it up slightly and21:17I add some base, so under slightly basic21:19conditions, these molecules will fall apart because you’ve21:23created conditions where the hydrogen bonding is no longer21:26favorable so they peel apart. If I had a beaker sitting on21:30the table here and it contained a million blue double stranded21:34DNA molecules and a million red double stranded DNA molecules21:37and I heated it up and added a little base,21:40I’d soon have four million individual strands just floating21:43around in the solution because I’ve broken up this hydrogen21:47bonding and the DNA molecules fall apart.21:49That’s called denaturing DNA. That tells you something about21:55the physical chemistry of the molecule;21:56that it’s these hydrogen bonds that hold the double strands and22:01I can break those down under certain conditions.22:05If I then put it back into its original condition,22:09lower the heat say, temperature back to body22:11temperature and reduce the pH down to seven again,22:15the molecules will re-nature. They will reform their natural22:19structure, and for DNA that means forming double helixes.22:23But they will do that in a very particular way,22:27in that only strands that exactly match will be able to22:32reform their native structure. A blue strand here will never22:38re-nature with a red strand because their sequences don’t22:43match exactly,22:4522:48but a complementary blue strand will always rematch with its22:53partner. Now this is the basis of a22:56physical chemistry process called hybridization.23:00It turns out that this is how we can identify specific DNA23:05sequences and how we can do things like DNA fingerprinting,23:11how we can clone molecules, DNA molecules from one organism23:15to another, rely very heavily on this principle of re-naturation23:21and hybridization. Hybridization simply means that23:25DNA will re-nature and form a stable double helix only with23:31its particular match, only with the hybrid that it is23:36perfectly complementary too.23:3823:43That’s something about the physical chemistry of DNA,23:47what it looks like, and how it behaves in the23:50simple sense. What I want to spend the rest23:53of the time doing is talking about some of the biological23:57properties of DNA. Again, I know this is something24:00that’s familiar to most of you and so indulge me just for the24:04rest of this lecture, I’ll go through it.24:06I want to try to hit the points that I think are important to24:09remember because they’re going to be concepts that come up24:11again and again throughout the course,24:13and I want to make sure that we’re on the same page.24:16This diagram at the top here is a very familiar one to24:20most of you, it’s sometimes called the central dogma of24:22molecular biology. It indicates how information24:26flows in cells and indicates a lot about the work that a cell24:30does in maintaining and recreating itself,24:33and maintaining its environment. That is, that the information24:36needed to operate a cell is stored in its DNA.24:40That information gets put into action through a process,24:43a biological process called transcription,24:47where particular regions of DNA are transcribed into RNA.24:51That RNA is made into proteins, and proteins are the working24:56molecules of the cell, they’re enzymes,24:58they’re structural molecules, they’re are proteins that exist25:01in the membrane that allow things to go in and out of the25:04cell, so really the working molecules25:06are the cell in every sense. RNA is converted into protein25:11by a process called translation. Here’s another picture of25:16it here, showing it in a little bit more detail,25:19that you have lots of DNA in each of the cells in your body25:22but you’re not using all that DNA at any one time.25:26Every cell in your body is only using a fraction of the DNA25:29that’s available to it. Cells in your pancreas,25:32for example, are making the protein insulin.25:35They’re making that because you need this protein insulin,25:38it’s a hormone, and it’s important for sugar25:40metabolism in your body. Those cells in your pancreas25:44are making insulin. That means the gene that25:48encodes insulin, the sequence of base pairs that25:51encode insulin. I’ll talk about what that25:55means, encoding insulin means in a minute, but there’s a gene25:59that tells your body what insulin looks like and that gets26:03transcribed but only in those cells that make insulin.26:08It gets converted into a protein, insulin,26:10only in those cells that are able to make the RNA that are26:13able to express the protein.26:1526:19Well, it turns out that proteins are essential in26:21driving this process too. In order to have DNA you have26:25to make DNA and your cells are continually making DNA inside26:31your body, through a process of DNA26:33synthesis and that synthesis is occurring because of the26:37presence of an enzyme, a protein called DNA polymerase.26:40In this same way, this process of transcription26:43which is occurring in cells throughout body all the time is26:46made possible by a protein called RNA polymerase.26:50It allows RNA to be made from a DNA template.26:55It’s not as simple as DNA going to RNA going to protein,26:59because proteins need to be present in order to make these27:04things happen as well. Let’s talk about DNA27:07synthesis for a minute. When a cell divides in your27:09body, when cells of your intestine divide,27:11when cells of your skin divide, and they’re doing this all the27:15time, in order for a cell to divide and form two daughter27:18cells–we’ll talk about that process next week–but in order27:22for that to happen the parent cell has to copy all of its DNA27:26in order to have enough DNA to pass on to two daughter cells.27:30It does that through a process of DNA synthesis.27:33What happens is the machinery of the cell, largely this27:38protein DNA polymerase, is able to open up the double27:43stranded DNA, to denature it locally,27:47exposing two strands which it then makes – allows it to make27:53copies of. What’s shown here is what’s27:56called a replication fork in DNA that’s undergoing synthesis.28:01The DNA molecule here has been spread apart,28:04opening up two single stranded DNA’s which have complementary28:09base sequences because they were double stranded DNA.28:13A new single stranded DNA is formed on each one of these open28:18single strands. So DNA is replicated using one28:24strand of the DNA as a template. The result of this process if28:31this replication went down the whole length of the DNA would be28:36to form two identical, double stranded DNA molecules.28:40Now the book talks in more detail about this and you can28:43read about it. Polymerase needs a primer and28:46that turns out to be important. A primer is a short RNA28:50sequence or DNA sequence that gets sort of the process of28:55replication jump started, and that’s just because of the28:59biological properties of DNA polymerase that that primer’s29:02needed. Synthesis always occurs in29:04one direction and that makes sense to you now because you29:07know there’s a directionality and the chemistry is different29:10going one way than the other and this DNA polymerase only works29:13on the chemistry going in one direction.29:15The correct complement is made because of these principles of29:20Watson-Crick base pairing that we talked about before.29:25It’s easy to know what nucleotide to put in each29:28position as you’re going along and polymerizing a new molecule.29:32Because this process occurs this way, if a parent cell29:35replicates its DNA and then passes them along to two29:38daughter cells, one of the daughter cells has29:41one strand from the parent, the dark blue strand here for29:46example, the other daughter cell has the29:48light blue strand, the complementary strand,29:50and each of the daughter cells has a newly synthesized piece of29:55DNA.29:5530:00That’s synthesis and that has to happen in order for cells to30:03replicate and cell replication is happening in your body all30:06the time. Transcription is also30:08happening. Certain segments of DNA are30:10being converted into RNA, and whereas in replication,30:14you have to copy the whole genome, the whole – all of the30:17chromosomes, all of the DNA contained in the chromosomes of30:21the cell in order to completely replicate it;30:24transcription only works on particular sequences of DNA.30:28The DNA that encodes the proteins that are important to30:32the life of that cell. A pancreas–cell in the30:35pancreas, for example, needs to make insulin and so30:38the gene for insulin is transcribed.30:40Transcription just means making a single stranded RNA30:47copy of a sequence of base pairs in a DNA.30:52I told you that that’s driven by a protein called RNA30:55polymerase. RNA polymerase is smart,30:57it knows where it needs to go in order to make the copy of RNA31:02that’s required. It operates in a similar31:06fashion to DNA polymerase in that it denatures locally or31:10opens up the double stranded DNA,31:12but it’s different in that it creates a new polymer from the31:18DNA template in the language of RNA,31:21using RNA nucleotides and not DNA nucleotides.31:25The end result of transcription is not double stranded DNA,31:29it’s single stranded RNA where the RNA that’s produced is31:34called messenger RNA. It’s the transcribed version of31:41DNA, and it’s the exact complement of a particular31:49region of DNA. Again, more details in your31:53book if you want to read that. Well, what I said is not31:56entirely true. That used to be the way that we31:59thought about it. DNA goes to RNA,32:02goes to protein, that’s it, and that is the way32:04it happens in simple organisms like bacteria.32:07In complex organisms like humans there’s another step that32:10we’re still only learning about now.32:12We know some parts of it, we don’t know all of it.32:14It’s very important in the biological operation of human32:19cells and that step is RNA splicing,32:22or processing of this RNA, single stranded RNA that’s32:27produced by transcription. We’re going to talk more about32:31this as we go through some specific examples of where RNA32:36processing is important. For now, just think about32:39modifying your picture of this sort of information flow through32:43a cell to include another step that RNA is produced by32:46transcription from DNA, double stranded DNA goes to a32:49single stranded RNA molecule, and that RNA is processed in32:54the cell in some way in order to form messenger RNA.32:58One of the forms of processing that happens,33:02that’s very important in human gene expression is that some of33:08the sections of the DNA molecule are not really necessary for33:13describing the protein. The regions that are necessary33:18for describing what the protein is like are called exons,33:21the regions that are not are called introns.33:24A section of DNA that is responsible for encoding a gene,33:27let’s say it’s the insulin gene for example,33:31might be some stretch of DNA on a certain chromosome inside your33:34cells, inside the cells of the pancreas.33:37If it was directly transcribed there’d be regions that are33:40important for making insulin and regions that are not.33:42Those regions that are not are spliced out during RNA33:47processing to form the mRNA transcript that’s used to make33:52the protein. Now there are other kinds33:55of processing that can happen to RNA as well, and again,33:58I said this is really still an emerging science,34:02but this is one that’s well known,34:04and you could imagine that it’s important.34:07If I want to clone a gene from a human, if I want to clone the34:10gene for human insulin, mean make many copies of the34:14gene that’s responsible for making insulin,34:17I need to know whether there are introns there or not.34:21If I’m going to make insulin from this I have to know that34:25I’ve got the introns spliced out correctly.34:29That’s something that will come up in the lecture tomorrow,34:32so remember that concept and that revision of this sort of34:36classical picture. I don’t want to go through34:39this in detail because I assume that you know it,34:41plus I think it’s a little bit easier to read and have some34:44time to digest, but this process of translation34:47or conversion of messenger RNA into a protein is a complicated34:51biological process that’s occurring all the time.34:54Before, we talked about how do you know what messenger RNA to34:58make, how do you know what RNA to copy from a DNA template?35:03Well you do that by this Watson-Crick base pairing,35:06so I know if I have (A)(C)(G)(C)(G)(A) I know what35:09messenger RNA to make from that because I have to satisfy these35:13base pairing rules. It’s more complicated in35:17making protein from an RNA strand and that complication is35:22called the genetic code. You know that messenger RNA is35:27read in three base units called codons, and so this particular35:33piece of messenger RNA is drawn in this cartoon in three base35:39pair units. That’s because every three base35:44pairs describes an amino acid in a protein.35:48While there are only four different bases that make up35:52either RNA or DNA, and so the complexity of an RNA35:56polymer is limited. It’s only got one of four36:00possible choices at each position.36:03There are more than 20 amino acids that make up the36:07biological polymers called proteins,36:09so there are 20 choices of each amino acid at a position on a36:17protein. Why are there three bases36:21in a codon? Because it takes three units36:25where there’s only four choices at each position to have at36:31least 20 unique combinations. How many combinations of codons36:37are there if there’s three bases and four possibilities at each36:41base? 4 x 4 x 4–possibilities,36:45because I could have (A)(G)(C)(U) here,36:51(A)(G)(C)(U), (A)(G)(C)(U) – 4 x 4 = 16.36:58If I only had two per codon I wouldn’t have enough.37:01I’d only have 16 possible two base sequences,37:04that’s not enough to specify over 20 amino acids.37:08If I have three, I have 16 x 4 or 64 possible37:13choices, way more than enough. That creates a problem in the37:19genetic code in that there’s 64 possible sequences but there’s37:23only 20 some amino acids, so each amino acid can be37:28specified by more than one codon.37:32There are combinations to spare. There are 64 combinations of37:38three bases and I only need to describe 20, so there’s37:42combinations to spare. If I look this table here37:47shows you how biological translation takes place whenever37:53a three base sequence is identified.37:56Say it’s (G)(C)(U), that specify an amino acid.38:01How would I know what amino acid that is?38:04Well, I could look up this table because somebody’s figured38:06it out for you. (G) in the first position,38:10(C) in the second position, (U) in the–(G)(C)(U) right38:17here is alanine and that’s the protein – that’s the amino acid38:24that’s in that position in the protein.38:30You could read through this sequence and you could figure38:34out what the sequence of amino acids would be.38:37The genetic code is said to be degenerate because I can read38:41in one direction. I can read (G)(C)(U) as38:44alanine, for example, from this table and if I see a38:47(G)(C)(U), I know it has to be alanine.38:50If I have the protein and I want to say what does the38:52messenger RNA that produced that protein looked like,38:55I can’t go backwards because there’s more than one39:01possibility for alanine, right?39:04There’s four of them right here (G)(C)(U), (G)(C)(C),39:08(G)(C)(A), (G)(C)(G) so I don’t know exactly what gene that came39:13from, I can’t read backwards. That has to do with the39:17statistics of this, right?39:19There’s just more sequences in a three unit codon than I need39:23for the amino acids. How does translation occur39:27biologically? As shown in this cartoon here,39:29again you don’t need to know the details of this,39:32but if you’re interested in knowing what’s the biological39:35bases of the genetic code this is it.39:37Inside cells in your body there are special RNA molecules called39:43transfer RNA. They are RNA molecules but they39:46have at some points in their life span, they have amino acids39:51attached to them. For example,39:54this transfer RNA has a unit here, (G)(A)(G) at one end of39:59the transfer RNA molecule. At the other end,40:04is attached the amino acid leucine.40:08And your cells making transfer RNA, if they make a transfer RNA40:13molecule that has (G)(A)(G) here, they only put leucine at40:18the other end. That’s the physical basis of40:22the genetic code because when a messenger RNA sequence is being40:27transcribed one base pair – one codon at a time,40:32when the sequence (C)(U)(C) appears in the messenger RNA,40:37that sequence (C)(U)(C) can only bind with one particular40:43three base complement, it has to be the complement40:48(G)(A)(G). This is the codon,40:51this is the anti-codon, there’s only one anti-codon40:55that matches this one and that anti-codon is always – occurs in41:00a molecule that has leucine attached to the other side.41:05Translation occurs by a special kind of polymerization41:10where these transfer RNA’s operate by Watson-Crick base41:15pairing. They bring into proximity an41:19amino acid so that instead of forming a new polymer of a41:25nucleic acid, a polymer of an amino acids is41:29formed. A polymer of amino acids is a41:32protein. Again, I’m just trying to41:34highlight things you already know a little bit about,41:37the book describes the details. This isn’t particularly41:41important for us to know here, but that messenger RNA gets41:46converted into a protein of a specific composition through a41:51biological process called translation is important.41:56The last thing I want to talk about today is control of41:59gene expression. Control of gene expression is a42:01very big topic and so I’m going to show you one cartoon to sort42:05of tell you that it is a big topic that’s really important.42:09Why is control of gene expression important?42:12Well I talked about it – earlier I’ve mentioned several42:15times all the cells in my body, all the cells in your body have42:20essentially the same genomic chromosomal DNA in their42:24nucleus. If you looked at cells in my42:27pancreas and cells in my brain, and cells in my skin they all42:29have the same DNA. But skin cells and brain cells42:32and pancreas cells aren’t doing the same things.42:36They don’t look the same, they don’t behave the same,42:38they don’t perform the same biological functions.42:40Why? Because brain cells and42:44pancreas cells are expressing different proteins.42:47All the cells in your body have the capability of making all the42:51proteins that you make, but they’re not all made in42:54every cell. Only cells of your pancreas42:57make insulin, for example.43:00Only cells in your brain make the enzymes that produce certain43:03neuron transmitters that are responsible for brain function.43:06How do cells in your brain know which proteins they ought43:09to be making, and how do cells in the43:11pancreas know which proteins they ought to be making?43:13They do that because they can control the expression of genes.43:20Gene expression, for us, will mean the same43:23thing as production of a particular protein.43:27When a gene gets expressed, that means its protein is43:32produced. When we talk about gene43:34expression than we’re talking about this whole sequence of43:37events I just described: transcription,43:39RNA processing, translation to make the43:44protein. All those things have to happen43:47in an orderly fashion, in enough quantity in order for43:51a particular cell to make a protein.43:54To make insulin, for example,43:55your cells of your pancreas have to be transcribing that43:58gene, it has to be processed,44:01has to be translated into the protein insulin.44:04But that’s not all, that protein insulin is made in44:08the form of a long polypeptide that not – that’s not always the44:14final version of the protein. In fact, for insulin,44:18it’s not the final version of the protein that comes out of44:20translation. There are more steps that have44:23to happen correctly in order for that insulin to become active.44:28Those steps are called post-translational44:31modifications. It’s a long word that just44:33means other chemistry that happens on the molecule after44:37translation. It turns out that the kinds of44:40post-translational modifications that human cells are able to do44:45are very complicated. You can do many44:47post-translational modifications;44:49your cells are capable of doing many post-translational44:51modifications. Bacteria, or simple organisms,44:54are not always capable of that. Now that’s going to be44:58important when we talk about making human gene – making human45:01proteins inside alternate hosts like bacteria,45:03that they can’t do all the things that your cells can do.45:08How is gene expression controlled?45:11It can be controlled in a variety of ways.45:13The most basic control is by controlling when transcription45:18happens. When transcription happens and45:20it turns out that there’s a whole biology associated with45:22this, including molecules that are45:24floating around inside your cells called transcription45:27factors, and their job– they are45:29molecules that are (that know) about particular genes and what45:33some of the sequences and are able to turn on those genes45:36inside cells, to make them transcribe.45:39It requires RNA processing to happen smoothly,45:42so if you can interfere with RNA processing you can stop a45:45gene from being expressed. If you can interfere with any45:49stage in RNA processing you can stop a gene from being expressed45:53and this is a very hot topic in molecular biology now and human45:57therapeutics. You’ve heard about RNA45:59interference, for example,46:01and that is the process of stopping this,46:03to stop a gene from being expressed.46:05For example, a gene that makes a cell46:07cancerous, I’d like to stop it from being expressed.46:10And you could interfere with a translation by degrading46:13messenger RNA, for example.46:15If you had a way to specifically chew up all the RNA46:17molecules that are responsible for making a particular protein,46:21you could stop it from being expressed even though your cell46:26is trying to make it. I want you to look at this46:29picture, read the little bit about gene – control of gene46:31expression – that’s in the book, know that it’s a big topic,46:34that we’re not going to talk about it except we’re going to46:37talk about some examples where control of gene expression can46:41be exploited in order to treat diseases,46:43for example. So I’ll see you on46:45Thursday.