1 00:00:33 --> 00:00:42 Good morning. Good morning. 2 00:00:42 --> 00:00:49 So, what I would like to do today is pick up on our basic theme of 3 00:00:49 --> 00:00:56 molecular biology. We've talked about DNA replication. 4 00:00:56 --> 00:01:03 The transcription of DNA into RNA, and the translation of RNA 5 00:01:03 --> 00:01:09 into protein. We discussed last time some of the variations between 6 00:01:09 --> 00:01:15 different types of organisms: viruses, prokaryotes, 7 00:01:15 --> 00:01:21 eukaryotes, with respect to the details of how they do that in 8 00:01:21 --> 00:01:27 general that bacteria have circular DNA chromosomes typically 9 00:01:27 --> 00:01:32 that eukaryotes have linear chromosomes, 10 00:01:32 --> 00:01:36 etc. What I'd like to talk about today is variation, 11 00:01:36 --> 00:01:41 but variation not between organisms but within an organism from time to 12 00:01:41 --> 00:01:46 time and place to place, namely, how it is that some genes or 13 00:01:46 --> 00:01:50 gene activities are turned on, on some occasions, and turned off on 14 00:01:50 --> 00:01:55 other occasions. This is, obviously, 15 00:01:55 --> 00:02:00 a very important problem to an organism, particularly 16 00:02:00 --> 00:02:03 to somebody like you who's a multi-cellular organism, 17 00:02:03 --> 00:02:06 and has the same DNA instruction set in all of your cells. 18 00:02:06 --> 00:02:09 It's obviously quite important to make sure that the same basic code 19 00:02:09 --> 00:02:13 is doing different things in different cells. 20 00:02:13 --> 00:02:16 It's important, also, to a bacterium to make sure that 21 00:02:16 --> 00:02:19 it's doing different things at different times, 22 00:02:19 --> 00:02:23 depending on its environment. So, I'm going to talk about a very 23 00:02:23 --> 00:02:26 particular system today as an illustration of how genes are 24 00:02:26 --> 00:02:30 regulated, but before we do that, let's 25 00:02:30 --> 00:02:35 Ask, where are the different places in this picture? 26 00:02:35 --> 00:02:40 DNA goes to DNA goes to RNA goes to protein, in which you might, 27 00:02:40 --> 00:02:45 in principle, regulate the activity of a gene. Could you regulate the 28 00:02:45 --> 00:02:50 activity of a gene by actually changing the DNA encoded in the 29 00:02:50 --> 00:02:55 genome? So, why not? Because what? It becomes a 30 00:02:55 --> 00:03:00 different gene. Yeah, that's just a definition. 31 00:03:00 --> 00:03:05 Why couldn't the cell just decide that I want this gene now to change 32 00:03:05 --> 00:03:10 in some way? Oh, I don't know, I'll alter the DNA 33 00:03:10 --> 00:03:16 sequence in some way. And, that'll make the gene work. 34 00:03:16 --> 00:03:21 Could that happen? Is that allowed? Yeah, it turns out to happen. 35 00:03:21 --> 00:03:27 It's not the most common thing, and it's not the thing they'll talk 36 00:03:27 --> 00:03:32 about in the textbooks a lot but you can actually do regulation. 37 00:03:32 --> 00:03:37 So, the levels of regulation are many, and one is actually at the 38 00:03:37 --> 00:03:42 level of DNA rearrangement. As we'll come to later in the 39 00:03:42 --> 00:03:47 course, for example, your immune system creates new, 40 00:03:47 --> 00:03:52 functional genes by rearranging locally some pieces of DNA, 41 00:03:52 --> 00:03:57 some bacteria, particularly infectious organisms control whether 42 00:03:57 --> 00:04:01 genes are turned on or off by actually going in there, 43 00:04:01 --> 00:04:04 and flipping around a piece of DNA in their chromosome. 44 00:04:04 --> 00:04:07 And, that's how they turn the gene on or off is they actually go in and 45 00:04:07 --> 00:04:10 change the genome. There's some protein that actually 46 00:04:10 --> 00:04:13 flips the orientation of a segment of DNA. Now, these are a little 47 00:04:13 --> 00:04:16 funky, and we're not going to talk a lot about them, 48 00:04:16 --> 00:04:19 but you should know, almost anything that can happen does 49 00:04:19 --> 00:04:22 happen and gets exploited in different ways by organisms. 50 00:04:22 --> 00:04:25 So, DNA rearrangement certainly happens. It's rare, 51 00:04:25 --> 00:04:29 but it's always cool when it happens. 52 00:04:29 --> 00:04:34 So, it's fun to look at. And, something like the immune 53 00:04:34 --> 00:04:40 system can't be dismissed as simply an oddity. That's an incredibly 54 00:04:40 --> 00:04:45 important thing. The most common form is at the 55 00:04:45 --> 00:04:51 level of transcriptional regulation, where whether or not a transcript 56 00:04:51 --> 00:04:57 gets made is how it's processed can be different. First off, 57 00:04:57 --> 00:05:02 the initiation of transcription that RNA polymerase should happen to 58 00:05:02 --> 00:05:06 sit down at this gene on this occasion and start transcribing it 59 00:05:06 --> 00:05:10 is a potentially regulatable (sic) step that maybe you're only going to 60 00:05:10 --> 00:05:14 turn on the gene for beta-globin and alpha-globin that together make the 61 00:05:14 --> 00:05:18 two components of hemoglobin, and you're only going to turn them 62 00:05:18 --> 00:05:22 on in red blood cells, or red blood cell precursors, 63 00:05:22 --> 00:05:26 and that could be done at the level of whether or not you make the 64 00:05:26 --> 00:05:30 message in the first place. That's one place it can be done. 65 00:05:30 --> 00:05:35 Another place is the splicing choices that you make. 66 00:05:35 --> 00:05:40 With respect to your message, you get this thing with a number of 67 00:05:40 --> 00:05:45 different potential exons, and you can regulate how this gene 68 00:05:45 --> 00:05:50 is used by deciding to splice it this way, and skip over that exon 69 00:05:50 --> 00:05:55 perhaps, or not skip over that exon. That alternative spicing is a 70 00:05:55 --> 00:06:00 powerful way to regulate. And then finally, you can also 71 00:06:00 --> 00:06:06 regulate at the level of mRNA stability. 72 00:06:06 --> 00:06:09 Stability means the persistence of the message, the degradation of the 73 00:06:09 --> 00:06:12 message. It could be that in certain cells, 74 00:06:12 --> 00:06:15 the message is protected so that it hangs around longer. 75 00:06:15 --> 00:06:18 And, in other cells, perhaps, it's unprotected and it's 76 00:06:18 --> 00:06:21 degraded very rapidly. If it's degraded very rapidly, 77 00:06:21 --> 00:06:24 it doesn't get a chance to make a protein or maybe it doesn't get to 78 00:06:24 --> 00:06:27 make too many copies of the protein. If it's persistent for a long time, 79 00:06:27 --> 00:06:30 it can make a lot of copies of protein. 80 00:06:30 --> 00:06:35 All of those things can and do occur. Then, of course, 81 00:06:35 --> 00:06:40 there is the regulation at the level of translation. 82 00:06:40 --> 00:06:46 Translation, if I give you an mRNA, is it automatically going to be 83 00:06:46 --> 00:06:51 translated? Maybe the cell has a way to sequester the RNA to ramp it 84 00:06:51 --> 00:06:57 up in some way so that it doesn't get to the ribosome under 85 00:06:57 --> 00:07:01 some conditions, and under other conditions it does 86 00:07:01 --> 00:07:05 get to the ribosome, or some ways to block in other 87 00:07:05 --> 00:07:08 manners than just sequestering it, but to physically block whether or 88 00:07:08 --> 00:07:12 not this message gets translated, what turns out that there's a 89 00:07:12 --> 00:07:15 tremendous amount of that. It's, again, not the most common, 90 00:07:15 --> 00:07:19 but we're learning, particularly over the last couple of years, 91 00:07:19 --> 00:07:22 that regulation of the translation of an mRNA is important. 92 00:07:22 --> 00:07:26 There are, although I won't talk about them at length, 93 00:07:26 --> 00:07:30 an exciting new set of genes called micro RNA's, 94 00:07:30 --> 00:07:34 teeny little RNAs that encode 21-22 base pair segments that are able to 95 00:07:34 --> 00:07:39 pair with a messenger RNA and interfere in some ways partially 96 00:07:39 --> 00:07:43 with its translatability. And so, by the number and the kinds 97 00:07:43 --> 00:07:48 of little micro RNAs that are there, organisms can tweak up or down how 98 00:07:48 --> 00:07:52 actively a particular message is being translated. 99 00:07:52 --> 00:07:57 So, the ability to regulate translation in a number of different 100 00:07:57 --> 00:08:01 ways is important. And then, of course, 101 00:08:01 --> 00:08:06 there's post-translational control. Once a protein is made, 102 00:08:06 --> 00:08:10 there's post-translational regulation that could happen. 103 00:08:10 --> 00:08:14 It could be that the protein is modified in some way. 104 00:08:14 --> 00:08:18 The proteins say completely inactive unless you put a phosphate 105 00:08:18 --> 00:08:22 group on it, and some enzyme comes along and puts a phosphate group on 106 00:08:22 --> 00:08:26 it. Or, it's inactive until you take off the phosphate group. 107 00:08:26 --> 00:08:30 All sorts of post-translational modifications can occur to proteins 108 00:08:30 --> 00:08:33 after the amino acid chain is made that can affect whether or not the 109 00:08:33 --> 00:08:37 protein is active. Every one of these is potentially a 110 00:08:37 --> 00:08:41 step by which an organism can regulate whether or not you have a 111 00:08:41 --> 00:08:45 certain biochemical activity present in a certain amount at a certain 112 00:08:45 --> 00:08:49 time. And, every one of these gets used. This is the thing about 113 00:08:49 --> 00:08:53 coming to a system that has been in the process of evolution for three 114 00:08:53 --> 00:08:57 and a half billion years is that even little differences can be 115 00:08:57 --> 00:09:01 fought over as competitive advantages, and can be fixed 116 00:09:01 --> 00:09:05 by an organism. So, if a tiny little thing began to 117 00:09:05 --> 00:09:10 help the organism slightly, it could reach fixation. And, 118 00:09:10 --> 00:09:15 you're coming along to this system, which has had about three and a half 119 00:09:15 --> 00:09:20 billion years of patches to the software code, 120 00:09:20 --> 00:09:25 and it's just got all sorts of layers and regulation piled on top 121 00:09:25 --> 00:09:30 of it. All of these things happen. But, what we think is the most 122 00:09:30 --> 00:09:35 important out of this whole collection is this guy. 123 00:09:35 --> 00:09:40 The fundamental place at which you're going to regulate whether or 124 00:09:40 --> 00:09:45 not you have the product of a gene is whether you bother to transcribe 125 00:09:45 --> 00:09:50 its RNA. But I do want to say because, yes? And, 126 00:09:50 --> 00:09:55 which exons you used and which aren't? Yeah, 127 00:09:55 --> 00:10:00 well, there are tissue-specific factors that are gene-specific 128 00:10:00 --> 00:10:03 that can influence that. And, surprisingly little is known 129 00:10:03 --> 00:10:06 about the details. There are a couple of cases where 130 00:10:06 --> 00:10:10 people know, but as you'd imagine, you actually need a regulatory 131 00:10:10 --> 00:10:13 system in that tissue to be able to decide to skip over that exon. 132 00:10:13 --> 00:10:17 And, the mechanics of that surprisingly are understood in very 133 00:10:17 --> 00:10:20 few cases. And, you might think that evolution 134 00:10:20 --> 00:10:23 wouldn't like to use that as the most common thing because you really 135 00:10:23 --> 00:10:27 do have to make a specialized thing to do that. So, that's what 136 00:10:27 --> 00:10:30 happens on these. That's one in particular where I 137 00:10:30 --> 00:10:33 think a tremendous amount of more work has to happen. 138 00:10:33 --> 00:10:36 mRNA stability, we understand some of it but not all the factors in 139 00:10:36 --> 00:10:39 this business. I was telling you about translation 140 00:10:39 --> 00:10:42 with these little micro-RNAs is stuff that's really only a few years 141 00:10:42 --> 00:10:45 old that people have come to understand. So, 142 00:10:45 --> 00:10:48 there's a lot to be understood about these things. I'm going to tell you 143 00:10:48 --> 00:10:51 about initiation of mRNAs, because it's the area where we know 144 00:10:51 --> 00:10:54 the most, and I think it'll give you a good idea of the general paradigm. 145 00:10:54 --> 00:10:57 But, any of you who want to go into this will find that there's a 146 00:10:57 --> 00:11:00 tremendous amount more to still be discovered about these things. 147 00:11:00 --> 00:11:05 So, the amount of protein that a cell might make varies wildly. 148 00:11:05 --> 00:11:10 Your red blood cells, 80% of your red blood cells, 149 00:11:10 --> 00:11:15 protein, is alpha or beta-globin. It's a huge amount. That's not 150 00:11:15 --> 00:11:21 true in any other cell in your body. So, we were talking about pretty 151 00:11:21 --> 00:11:26 significant ranges of difference as to how much protein is made. 152 00:11:26 --> 00:11:31 How do things like that happen? Well, I'm going to describe the 153 00:11:31 --> 00:11:37 simplest and classic case of gene regulation and bacteria, 154 00:11:37 --> 00:11:43 and in particular, the famous lack operon of E coli. 155 00:11:43 --> 00:11:49 So, this was the first case in which regulation was ever really 156 00:11:49 --> 00:11:55 worked out, and it stands today as a very good paradigm of how 157 00:11:55 --> 00:12:00 regulation works. E coli, in order to grow, 158 00:12:00 --> 00:12:05 needs a carbon source. In particular, E coli is fond of sugar. 159 00:12:05 --> 00:12:10 It would like to have a sugar to grow on. Given a choice, 160 00:12:10 --> 00:12:15 what's E coli's favorite sugar? It's glucose, right, because we 161 00:12:15 --> 00:12:20 have the whole cycle of glucose. The whole pathway of glucose goes 162 00:12:20 --> 00:12:25 to pyruvate, which we've talked about, and glucose is the preferred 163 00:12:25 --> 00:12:30 sugar to go into that pathway, OK, of glycolysis. 164 00:12:30 --> 00:12:36 Glycolysis: the breakdown of glucose. But, suppose there's no glucose 165 00:12:36 --> 00:12:42 available. Is E coli willing to have a different sugar? 166 00:12:42 --> 00:12:48 Sure, because E coli's not stupid. If it were to refuse another sugar, 167 00:12:48 --> 00:12:54 it wouldn't be able to grow. So, it has a variety of pathways that will 168 00:12:54 --> 00:13:00 shunt other sugars to glucose, which will then allow you to go 169 00:13:00 --> 00:13:08 through glycolysis, etc. Now, given a choice, 170 00:13:08 --> 00:13:16 it would prefer to use the glucose. But if not, suppose you gave it 171 00:13:16 --> 00:13:24 lactose. Lactose is a disaccharide. It's milk sugar, and I'll just 172 00:13:24 --> 00:13:32 briefly sketch, so lactose is a disaccharide where 173 00:13:32 --> 00:13:40 you've got a glucose and a galactose. 174 00:13:40 --> 00:13:50 Glucose plus galactose equals lactose. So, if E coli is given 175 00:13:50 --> 00:14:00 galactose, it is able to break it down into glucose plus galactose. 176 00:14:00 --> 00:14:08 And it does that by a particular enzyme called beta galactosidase, 177 00:14:08 --> 00:14:17 which breaks down glactosides. And, it'll give you galactose plus 178 00:14:17 --> 00:14:25 glucose. How much beta-galactosidase does an E coli 179 00:14:25 --> 00:14:32 cell have around? Sorry? None? But how does it do this? 180 00:14:32 --> 00:14:38 When it needs it, it'll synthesize it. When it needs it, 181 00:14:38 --> 00:14:43 like, there's no glucose and there's a lot of galactose around, 182 00:14:43 --> 00:14:49 how much of it will there be? A lot. It turns out that in 183 00:14:49 --> 00:14:54 circumstances where E coli is dependent on galactose as its fuel, 184 00:14:54 --> 00:15:00 something like 10% of total protein 185 00:15:00 --> 00:15:06 can be beta-gal under the circumstances when you have 186 00:15:06 --> 00:15:13 galactose but no glucose. Sorry? Sorry, when you have 187 00:15:13 --> 00:15:19 lactose but no glucose. Thank you. So, when you have 188 00:15:19 --> 00:15:26 lactose but no glucose, E coli has 10% of its protein weight 189 00:15:26 --> 00:15:33 as beta-galactosidase. Wow. But when you have glucose 190 00:15:33 --> 00:15:39 around or you don't have lactose around, you have very little. 191 00:15:39 --> 00:15:45 It could be almost none, trace amounts. So, why do this? 192 00:15:45 --> 00:15:51 Why not, for example, just have a far more reasonable some compromise? 193 00:15:51 --> 00:15:57 Like, let's always just have 1% of beta-galactosidase. 194 00:15:57 --> 00:16:04 Why do we need the 0-10%? 10%'s actually extremely high. 195 00:16:04 --> 00:16:09 So what. It's a good insurance policy. So, if I only have 196 00:16:09 --> 00:16:15 galactose, I need more. Well, I mean, 1% will still digest 197 00:16:15 --> 00:16:21 it. I'll still do it. What's the problem? Sorry? 198 00:16:21 --> 00:16:27 So what, I do it at a slower rate. Life's long. Why not? Ah, it has 199 00:16:27 --> 00:16:31 to compete. So, if the cell to the left had a 200 00:16:31 --> 00:16:35 mutation that got it to produce four times as much, 201 00:16:35 --> 00:16:38 then it would soak up the lactose in the environment, 202 00:16:38 --> 00:16:42 grow faster, etc. etc., and we could have competed. 203 00:16:42 --> 00:16:45 So, these little tuning mutations have a huge effect amongst this 204 00:16:45 --> 00:16:49 competing population of bacteria. And so, if E coli currently thinks 205 00:16:49 --> 00:16:53 that it's really good to have almost non at sometimes and 10% at other 206 00:16:53 --> 00:16:56 times, you can bet that it's worked that out through the product of 207 00:16:56 --> 00:17:00 pretty rigorous competition, that it doesn't want to waste the 208 00:17:00 --> 00:17:03 energy making this when you don't need it, and that when you do need 209 00:17:03 --> 00:17:07 it, you really have to compete hard by growing as fast as you can when 210 00:17:07 --> 00:17:11 you have that lactose around. OK. So, how does it actually get 211 00:17:11 --> 00:17:16 the lactose, sorry, keep me honest on lactose versus 212 00:17:16 --> 00:17:22 galactose, into the cell? It turns out that it also has 213 00:17:22 --> 00:17:27 another gene product, another protein, which is a lactose 214 00:17:27 --> 00:17:32 permease. And, any guesses as to what a 215 00:17:32 --> 00:17:38 lactose permease does? It makes the cell permeable to 216 00:17:38 --> 00:17:43 lactose, right, good. So, the lactose can get into 217 00:17:43 --> 00:17:49 the cell, and then beta-gal can break it down into galactose plus 218 00:17:49 --> 00:17:54 glucose. These two things, in fact, both get regulated, 219 00:17:54 --> 00:18:00 beta-gal and this lactose permease. So, how does it work? 220 00:18:00 --> 00:18:07 Let's take a look now at the structure of the lack operon. 221 00:18:07 --> 00:18:14 So, I mentioned briefly last time, what's an operon? Remember we said 222 00:18:14 --> 00:18:21 that in bacteria, you often made a transcript that had 223 00:18:21 --> 00:18:29 multiple proteins that were encoded on it. 224 00:18:29 --> 00:18:33 A single mRNA could get made, and multiple starts for translation 225 00:18:33 --> 00:18:37 could occur, and you could make multiple proteins. 226 00:18:37 --> 00:18:41 And, this would be a good thing if you wanted to make a bunch of 227 00:18:41 --> 00:18:45 proteins that were a part of the same biochemical pathway. 228 00:18:45 --> 00:18:49 Such an object, a regulated piece of DNA that makes a transcript 229 00:18:49 --> 00:18:53 encoding multiple polypeptides is called an operon because they're 230 00:18:53 --> 00:18:57 operated together. So, let's take a look here at the 231 00:18:57 --> 00:19:03 lack operon. I said there was a promoter. 232 00:19:03 --> 00:19:09 Here is a promoter for the operon, and we'll call it P lack, promoter 233 00:19:09 --> 00:19:16 for the lack operon. Here is the first gene that is 234 00:19:16 --> 00:19:23 encoded. So, the message will start here, actually about here, 235 00:19:23 --> 00:19:30 and start going off. And, the first gene is given the name lack Z. 236 00:19:30 --> 00:19:34 It happens to encode beta-galactosidase enzyme. 237 00:19:34 --> 00:19:38 Remember, they did a mutant hunt, and when they did the mutant hunt, 238 00:19:38 --> 00:19:42 they didn't know what each gene was as they isolated mutants. 239 00:19:42 --> 00:19:46 So, they just gave them names of letters. And so, 240 00:19:46 --> 00:19:50 it's called lack Z. And, everybody in molecular biology 241 00:19:50 --> 00:19:54 knows this is the lack Z gene, although Z has nothing to do with 242 00:19:54 --> 00:19:58 beta-galactosidase. It was just the letter given to it. 243 00:19:58 --> 00:20:02 But, it's stuck. Next is lack Y. 244 00:20:02 --> 00:20:07 And, that encodes the permease. And, there is also lack A, which 245 00:20:07 --> 00:20:12 encodes a transacetylase, and as far as I'm concerned you can 246 00:20:12 --> 00:20:17 forget about it. OK, but I just mentioned that it is 247 00:20:17 --> 00:20:22 there, and it actually does make three polypeptides. 248 00:20:22 --> 00:20:27 We won't worry about it, OK, but it does make a 249 00:20:27 --> 00:20:32 transacetylase, OK? But it won't figure in what we're 250 00:20:32 --> 00:20:36 going to talk about, and actually remarkably little is 251 00:20:36 --> 00:20:41 known about the transacetylase. There's also one other gene I need 252 00:20:41 --> 00:20:46 to talk about, and that's over here, 253 00:20:46 --> 00:20:50 and that's called lack I. And, it too has a promoter, 254 00:20:50 --> 00:20:55 which we can call PI, for the promoter for lack I. 255 00:20:55 --> 00:21:00 And, this encodes a very interesting protein. 256 00:21:00 --> 00:21:05 So, we get here one message encoding one polypeptide here. 257 00:21:05 --> 00:21:11 This mRNA encodes one polypeptide. It is monocystronic. This guy here 258 00:21:11 --> 00:21:17 is a polycystronic message. It has multiple cystrons, which is 259 00:21:17 --> 00:21:23 the dusty old name for these regions that were translated into distinct 260 00:21:23 --> 00:21:29 proteins. And so, that's that mRNA. 261 00:21:29 --> 00:21:40 So, lack I, this encodes a very interesting protein, 262 00:21:40 --> 00:21:52 which is called the lack repressor. The lack repressor, actually I'll 263 00:21:52 --> 00:22:04 bring this down a moment, is not an enzyme. 264 00:22:04 --> 00:22:10 It's not a self-surface channel for putting in galactose. 265 00:22:10 --> 00:22:17 It is a DNA binding protein. It binds to DNA. But, it's not a 266 00:22:17 --> 00:22:23 nonspecific DNA binding protein that binds to any old DNA. 267 00:22:23 --> 00:22:30 It has a sequence-specific preference. 268 00:22:30 --> 00:22:35 It's a protein that has a particular confirmation, a particular shape, 269 00:22:35 --> 00:22:40 a particular set of amino acids sticking out, that it combined into 270 00:22:40 --> 00:22:46 the major groove of DNA in a sequence-specific fashion such that 271 00:22:46 --> 00:22:51 it particularly likes to recognize a certain sequence of nucleotides and 272 00:22:51 --> 00:22:57 binds there. Where is the specific sequence of nucleotides where this 273 00:22:57 --> 00:23:03 guy likes to bind? It so happens that it's there. 274 00:23:03 --> 00:23:11 And this is called the operator sequence or the operator site. 275 00:23:11 --> 00:23:18 So, this protein likes to go and bind there. Now, 276 00:23:18 --> 00:23:26 I've drawn this, by the way, so that this operator site is 277 00:23:26 --> 00:23:34 actually right overlapping the promoter site. 278 00:23:34 --> 00:23:40 Who likes to bind at the promoter site? RNA polymerase. 279 00:23:40 --> 00:23:46 What's going to happen if the lack repressor protein is sitting there? 280 00:23:46 --> 00:23:52 RNA polymerase can't bind. It's just physically, 281 00:23:52 --> 00:23:58 blocked from binding. So, let's examine some cases here. 282 00:23:58 --> 00:24:06 Let's suppose that we look at here 283 00:24:06 --> 00:24:14 at our gene. We've got our promoter, P lack. We've got the operator site 284 00:24:14 --> 00:24:22 here. We've got the lack Z gene here, and we've got the lack 285 00:24:22 --> 00:24:31 repressor, lack I, the repressor sitting there. 286 00:24:31 --> 00:24:38 Polymerase tries to come along to this, and it's blocked. 287 00:24:38 --> 00:24:45 So, what will happen in terms of the transcription of the lack 288 00:24:45 --> 00:24:52 operon: no mRNA. So, that's great. 289 00:24:52 --> 00:24:59 So, we've solved one problem right off the bat. 290 00:24:59 --> 00:25:03 We want to be sure that sometimes there's going to be no mRNA made. 291 00:25:03 --> 00:25:07 This way, we're not going to waste any metabolic energy, 292 00:25:07 --> 00:25:11 making beta-galactosidase. Are we done? No? Why not. 293 00:25:11 --> 00:25:16 We've got to sometimes make beta-galactosidase. 294 00:25:16 --> 00:25:20 So, we've got to get that repressor off there. Well, 295 00:25:20 --> 00:25:24 how is the repressor going to come off there? When do we want the 296 00:25:24 --> 00:25:29 repressor off there: when there's lactose present. 297 00:25:29 --> 00:25:34 So, somehow we need to build some kind of an elaborate sensory 298 00:25:34 --> 00:25:39 mechanism that is able to tell when lactose is present, 299 00:25:39 --> 00:25:44 and send a signal to the repressor protein saying, 300 00:25:44 --> 00:25:49 hey, lactose is around. The signal gets transmitted all the 301 00:25:49 --> 00:25:55 way to the repressor protein, and the repressor protein comes off. 302 00:25:55 --> 00:26:00 What kind of an elaborate sensory mechanism might be built? 303 00:26:00 --> 00:26:05 Use lactose as what? So, this is actually pretty simple. 304 00:26:05 --> 00:26:11 You're saying just take lactose, and you want lactose to be its own 305 00:26:11 --> 00:26:16 signal? So, if lactose were to just bind to the repressor, 306 00:26:16 --> 00:26:21 the repressor might then know that there was lactose around. 307 00:26:21 --> 00:26:27 Well, what would it do if lactose bound to it? Sorry? Why 308 00:26:27 --> 00:26:33 would it fall off? Yep. More interested in the lactose. 309 00:26:33 --> 00:26:39 So, if you're suggestion, this is good. I like the design 310 00:26:39 --> 00:26:45 work going on here. The suggestion is that if lactose 311 00:26:45 --> 00:26:51 binds to this here, binds to our repressor, 312 00:26:51 --> 00:26:57 it's going to fall off because it's more interested in lactose 313 00:26:57 --> 00:27:03 than in the DNA. Now, how is the interest actually 314 00:27:03 --> 00:27:07 conveyed into something material? Because the actual level of 315 00:27:07 --> 00:27:11 cognitive like or dislike for DNA on the part of this polypeptide is 316 00:27:11 --> 00:27:15 unclear, you may be anthropomorphizing slightly with 317 00:27:15 --> 00:27:19 regard to this polypeptide chain. So, mechanistically, what's going 318 00:27:19 --> 00:27:23 to happen? Shape. Yes, shape? Change confirmation, 319 00:27:23 --> 00:27:27 the binding act, the act of binding lactose creates some energy, 320 00:27:27 --> 00:27:31 may change the shape of the protein, 321 00:27:31 --> 00:27:35 and that shape of the protein may, in the process of wiggling around to 322 00:27:35 --> 00:27:40 bind lactose may de-wiggle some other part of it that now no longer 323 00:27:40 --> 00:27:44 binds so well to DNA. That is exactly what happens. 324 00:27:44 --> 00:27:49 Good job. So, you guys have designed, in fact, 325 00:27:49 --> 00:27:54 what really happens. What happens is what's called an 326 00:27:54 --> 00:27:58 allosteric change. It just means other shape. 327 00:27:58 --> 00:28:03 So, it just changes its shape, that it changes shape on binding of 328 00:28:03 --> 00:28:10 lactose. And it falls off because it's less 329 00:28:10 --> 00:28:18 suitable for binding this particular DNA sequence when it's bound to 330 00:28:18 --> 00:28:26 lactose there. So, in this case, 331 00:28:26 --> 00:28:34 in the presence of lactose, lack I does not bind. 332 00:28:34 --> 00:28:44 And, the lack operon is transcribed. Yes? Uh-oh. OK, all right 333 00:28:44 --> 00:28:54 designers, here we've got a problem. You have such a cool system, 334 00:28:54 --> 00:29:03 right? You were going to sense lactose. 335 00:29:03 --> 00:29:10 Lactose was going to bind to the lack repressor, 336 00:29:10 --> 00:29:17 change its confirmation falloff: uh-oh. But, as you point out, 337 00:29:17 --> 00:29:25 how's it going to get any lactose, because there's not a lactose 338 00:29:25 --> 00:29:32 permease because the lactose permease is made by 339 00:29:32 --> 00:29:37 the same operon. So, what if, in fact, 340 00:29:37 --> 00:29:40 instead of getting one of these DOD mill speck kind of things of some 341 00:29:40 --> 00:29:43 repressor that is absolutely so tight that it never falls off under 342 00:29:43 --> 00:29:47 any circumstances, what if we build a slightly sloppy 343 00:29:47 --> 00:29:50 repressor that occasionally falls off, and occasionally allows 344 00:29:50 --> 00:29:53 transcription of the lack operon? Then, we'll have some trace 345 00:29:53 --> 00:29:56 quantities of permease around. With a little bit of permease 346 00:29:56 --> 00:30:00 around, a little lactose will get in. 347 00:30:00 --> 00:30:04 And, as long as even a little lactose gets in, 348 00:30:04 --> 00:30:08 it'll now shift the equilibrium so that the repressor is off more, 349 00:30:08 --> 00:30:12 and of course that will make more permease, and shift, 350 00:30:12 --> 00:30:16 and shift, and shift, and shift. So, as long as it's not 351 00:30:16 --> 00:30:21 so perfectly engineered as to have nothing being transcribed, 352 00:30:21 --> 00:30:25 so no mRNA is really very little mRNA. See, this is what's so good, 353 00:30:25 --> 00:30:29 I think, about having MIT students learn this stuff because there are 354 00:30:29 --> 00:30:33 all sorts of wonderful design principles here about how 355 00:30:33 --> 00:30:38 you build systems. And, I think this is just a very 356 00:30:38 --> 00:30:43 good example of how you build a system like this. 357 00:30:43 --> 00:30:48 Now, all right, so we now have the ability to have lack on and lack off, 358 00:30:48 --> 00:30:52 and that is lack off, mostly off because of your permease 359 00:30:52 --> 00:30:57 problem: very good. Now, let's take a little digression 360 00:30:57 --> 00:31:02 about, how do we know this? This kind of reasoning, 361 00:31:02 --> 00:31:06 I've now told you the answer. But let's actually take a look at 362 00:31:06 --> 00:31:10 understanding the evidence that lets you conclude this. 363 00:31:10 --> 00:31:14 So, in order to do this, and this is the famous work in 364 00:31:14 --> 00:31:18 molecular biology of Jacobin Manoux in the late '50s for which they won 365 00:31:18 --> 00:31:22 a Nobel Prize, they wanted to collect some mutants. 366 00:31:22 --> 00:31:26 Remember, this is before the time of DNA sequence or anything like 367 00:31:26 --> 00:31:30 that, and wanted to collect mutants that affected this process. 368 00:31:30 --> 00:31:37 So, in order to collect mutants that screwed up the regulation, 369 00:31:37 --> 00:31:45 they knew that beta-galactosidase was produced in much higher quantity 370 00:31:45 --> 00:31:53 if lactose was around. The difficulty with that was that 371 00:31:53 --> 00:32:01 wild type E coli, when you had no lactose would 372 00:32:01 --> 00:32:07 produce very little beta-gal, one unit of beta-gal, 373 00:32:07 --> 00:32:11 and in the presence of lactose, would produce a lot, let's call it 1, 374 00:32:11 --> 00:32:16 00 units of beta-gal. But, the problem with playing 375 00:32:16 --> 00:32:20 around with this is lactose is serving two different roles. 376 00:32:20 --> 00:32:25 Lactose is both the inducer of the expression of the gene by virtue of 377 00:32:25 --> 00:32:30 binding to the repressor, etc., etc. 378 00:32:30 --> 00:32:34 But, it's also the substrate for the enzyme because as beta-galactosidase 379 00:32:34 --> 00:32:38 gets made, it breaks down the lactose. So, there's less lactose 380 00:32:38 --> 00:32:43 in binding, and if you wanted to really study the regulatory controls, 381 00:32:43 --> 00:32:47 you have the problem that the thing that's inducing the gene by binding 382 00:32:47 --> 00:32:52 to the repressor is the thing that's getting destroyed by the product of 383 00:32:52 --> 00:32:56 the gene. So, it's going to make the kinetics of 384 00:32:56 --> 00:33:01 studying such a process really messy. It would be very nice if you could 385 00:33:01 --> 00:33:05 make a form of lactose that could induce beta-galactosidase by binding 386 00:33:05 --> 00:33:10 to the repressor, but wasn't itself digested. 387 00:33:10 --> 00:33:17 Chemically, in fact, you can do that. Chemically, 388 00:33:17 --> 00:33:24 it's possible to make a molecule called IPTG, which is a 389 00:33:24 --> 00:33:32 galactoside analog. And, what it does is this molecule 390 00:33:32 --> 00:33:41 here which I'll just sketch very quickly here, it's a sulfur there, 391 00:33:41 --> 00:33:50 and you can see vaguely similar, this is able to be an inducer. 392 00:33:50 --> 00:33:59 It'll induce beta-gal, but not a substrate. It won't get digested. 393 00:33:59 --> 00:34:04 So, it'll stick around as long as you want. It's also very convenient 394 00:34:04 --> 00:34:09 to use a molecule that was developed called ex-gal. 395 00:34:09 --> 00:34:15 Ex-gal again has a sugar moiety, and then it also has this kind of a 396 00:34:15 --> 00:34:20 funny double ring here, which is a chlorine, and a bromine, 397 00:34:20 --> 00:34:25 and etc. And, this guy here is not an inducer. It's not capable of 398 00:34:25 --> 00:34:31 being induced, of inducing beta-galactosidase 399 00:34:31 --> 00:34:37 expression. But, it is a substrate. 400 00:34:37 --> 00:34:43 It will be broken down by the enzyme, and rather neatly when it's 401 00:34:43 --> 00:34:49 broken down it turns blue. These two chemicals turned out to 402 00:34:49 --> 00:34:55 be very handy in trying to work out the regulation of the 403 00:34:55 --> 00:35:00 lack operon. So, if I, instead of adding lactose, 404 00:35:00 --> 00:35:06 if I think about adding IPTG, my inducer, when I add IPTG I'm going 405 00:35:06 --> 00:35:12 to get beta-gal produced. When I don't have IPTG, I won't 406 00:35:12 --> 00:35:18 produce beta-gal. But then I don't have a problem of 407 00:35:18 --> 00:35:24 this getting used up. So now, what kind of a mutant might 408 00:35:24 --> 00:35:29 I look for? I might look for a mutant that even 409 00:35:29 --> 00:35:34 in the absence of the inducer, IPTG, still produces a lot of 410 00:35:34 --> 00:35:39 beta-gal. Now, I can also look for mutants that no 411 00:35:39 --> 00:35:44 matter what never produce beta-gal, right? But, what would they likely 412 00:35:44 --> 00:35:50 be? They'd likely be structural mutations affecting the coding 413 00:35:50 --> 00:35:55 sequence of beta-gal, right? Those will happen. 414 00:35:55 --> 00:36:00 I can collect mutations that cause the E coli never to 415 00:36:00 --> 00:36:05 produce beta-gal. But that's not as interesting as 416 00:36:05 --> 00:36:11 collecting mutations that block the repression that cause beta-gal to be 417 00:36:11 --> 00:36:16 produced all of the time. So, how would I find such a mutant? 418 00:36:16 --> 00:36:22 I want to find a mutant that's producing a lot of beta-gal even 419 00:36:22 --> 00:36:27 when there's no IPTG. So, let's place some E coli on a 420 00:36:27 --> 00:36:33 plate. Should we put IPTG on a plate? No, so no IPTG. 421 00:36:33 --> 00:36:37 What do I look for? How do I tell whether or not any of 422 00:36:37 --> 00:36:42 these guys here is producing a lot of beta-gal? Yep? 423 00:36:42 --> 00:36:46 So, no IPTG, but put on ex-gal, and if anybody's producing a lot of 424 00:36:46 --> 00:36:51 beta-gal, what happens? They turn blue: very easy to go 425 00:36:51 --> 00:36:55 through lots of E coli like that looking for something blue. 426 00:36:55 --> 00:37:00 And so, lots of mutants were collected that were blue. 427 00:37:00 --> 00:37:05 And, these chemicals are still used today. They're routinely used in 428 00:37:05 --> 00:37:10 labs, ex-gal and stuff like that, making bugs turn blue because this 429 00:37:10 --> 00:37:15 has turned out to be such a well-studied system that we use it 430 00:37:15 --> 00:37:20 for a lot of things. So, mutants were found that were 431 00:37:20 --> 00:37:25 constituative. So, mutants were found that were 432 00:37:25 --> 00:37:30 constituative mutants. Constituative mutants: meaning 433 00:37:30 --> 00:37:35 expressing all the time, no longer regulated, so, 434 00:37:35 --> 00:37:40 characterizing these constituative mutants. 435 00:37:40 --> 00:37:44 It turns out that they fell into two different classes of constituative 436 00:37:44 --> 00:37:48 mutants. If we had enough time, and you could read the papers and 437 00:37:48 --> 00:37:52 all, what I would do is give you the descriptions that Jacobin Maneaux 438 00:37:52 --> 00:37:56 had of these funny mutants which they'd isolated and were trying to 439 00:37:56 --> 00:38:00 characterize, and how to puzzle out what was going on. 440 00:38:00 --> 00:38:04 But, it's complicated and hard, and makes your head hurt if you 441 00:38:04 --> 00:38:08 don't know what the answer is. So, I'm going to first tell you the 442 00:38:08 --> 00:38:12 answer of what's going on, and then sort of see how you would 443 00:38:12 --> 00:38:17 know that this was the case. But, imagine that you didn't know 444 00:38:17 --> 00:38:21 this answer, and had to puzzle this out from the data. 445 00:38:21 --> 00:38:25 So, suppose we had, so if there were going to be two 446 00:38:25 --> 00:38:30 kinds of mutants: mutant number one are operator constituents. 447 00:38:30 --> 00:38:38 They have a defective operator sequence. Mutations have occurred 448 00:38:38 --> 00:38:46 at the operator site. Mutant number two have a defective 449 00:38:46 --> 00:38:54 repressor protein, the gene for the repressor protein. 450 00:38:54 --> 00:39:00 How can I tell the difference? 451 00:39:00 --> 00:39:04 So, I could have a problem in my operator site. 452 00:39:04 --> 00:39:08 What would be the problem with the operator site? 453 00:39:08 --> 00:39:12 Some mutation to the sequence causes the repressor not to bind 454 00:39:12 --> 00:39:16 there anymore, OK? So, a defective operator site 455 00:39:16 --> 00:39:20 doesn't bind repressors. Defective repressor, the operator 456 00:39:20 --> 00:39:24 site is just fine, but I don't have a repressor to bind 457 00:39:24 --> 00:39:28 at it. So how do I tell the difference? One way to tell the 458 00:39:28 --> 00:39:32 difference is to begin crossing the mutants together to wild type, 459 00:39:32 --> 00:39:36 and asking, are they dominant or recessive, or things like that? 460 00:39:36 --> 00:39:39 Now, here's a little problem. E Coli is not a diploid, so you 461 00:39:39 --> 00:39:43 can't cross together two E colis and make a diploid E coli, 462 00:39:43 --> 00:39:46 right? It's a prokaryote. It only has one genome. But, 463 00:39:46 --> 00:39:50 it turns out that you can make temporary diploids, 464 00:39:50 --> 00:39:53 partial diploids out of E coli because it turns out you can mate 465 00:39:53 --> 00:39:57 bacteria. Bacteria, which have a bacterial chromosome 466 00:39:57 --> 00:40:01 here also engage in sex and in the course of bacterial sex, 467 00:40:01 --> 00:40:05 plasmids can be transferred called, for example, an F factor, is able to 468 00:40:05 --> 00:40:10 be transferred from another bacteria. And, through the wonders of partial 469 00:40:10 --> 00:40:15 merodiploid, you can temporarily get E colis, or you can permanently get 470 00:40:15 --> 00:40:20 E colis, that are partially diploid. So, you can do what I'm about to 471 00:40:20 --> 00:40:25 say. But, in case you were worried about my writing diploid genotypes 472 00:40:25 --> 00:40:30 for E coli, you can actually do this. 473 00:40:30 --> 00:40:36 You can make partial diploids. So, let's try out a genotype here. 474 00:40:36 --> 00:40:43 Suppose the repressor is a wild type, the operator is wild type, 475 00:40:43 --> 00:40:49 and the lack Z gene is wild type. And, suppose I have no IPTG, I'm 476 00:40:49 --> 00:40:56 un-induced. I have one unit of beta-gal. When I add my inducer, 477 00:40:56 --> 00:41:03 what happens? I get 1,000 units of beta-gal. 478 00:41:03 --> 00:41:07 Now, suppose I would have an operator constituative mutation. 479 00:41:07 --> 00:41:12 Then, the operator site is defective. It doesn't bind the 480 00:41:12 --> 00:41:17 repressor. Beta-gal is going to be expressed all the time, 481 00:41:17 --> 00:41:22 even in the absence. All right, well that was, of course, what we 482 00:41:22 --> 00:41:27 selected for. Now, suppose I made the following diploid. 483 00:41:27 --> 00:41:32 I plus, O plus, Z plus, over I plus, 484 00:41:32 --> 00:41:38 O constituative, Z plus. So, here's my diploid. What would 485 00:41:38 --> 00:41:44 be the phenotype? So, in other words, 486 00:41:44 --> 00:41:50 one of the chromosomes has an operator problem. 487 00:41:50 --> 00:41:56 Well, that means that this chromosome here is always going to 488 00:41:56 --> 00:42:02 be constituatively expressing beta-gal. 489 00:42:02 --> 00:42:06 But, what about this chromosome here? It won't. So, 490 00:42:06 --> 00:42:10 this would be about 1, 01, give or take, because it's got 491 00:42:10 --> 00:42:14 one chromosome doing that and one chromosome doing this, 492 00:42:14 --> 00:42:18 and this one would be about 2, 00. Now, that quantitative 493 00:42:18 --> 00:42:22 difference doesn't matter a lot. What you really saw when you did 494 00:42:22 --> 00:42:26 the molecular biology was that when you had one copy of the operator 495 00:42:26 --> 00:42:30 constituative mutation, you still got a lot of beta-gal here 496 00:42:30 --> 00:42:36 even in the absence of IPTG. So, that operator constituative site 497 00:42:36 --> 00:42:44 looked like it was dominant to this plus site here. 498 00:42:44 --> 00:42:52 But now, let's try this one here. I plus, O plus, Z plus, over I plus, 499 00:42:52 --> 00:43:00 operator constituative, Z minus. What happens then? 500 00:43:00 --> 00:43:05 This operator constituative site allows constant transcription of 501 00:43:05 --> 00:43:10 this particular copy. But, can this particular copy make 502 00:43:10 --> 00:43:15 a working, functional beta-gal? No. So, this looks, when you do 503 00:43:15 --> 00:43:20 your genetic crosses, you find that the operator 504 00:43:20 --> 00:43:25 constituative, now, if I reverse these here, 505 00:43:25 --> 00:43:30 suppose I reverse these, I plus, O plus, Z minus, I plus, O 506 00:43:30 --> 00:43:35 constituative, Z plus, same genotypes, 507 00:43:35 --> 00:43:40 right, except that I flipped which chromosome these are on. 508 00:43:40 --> 00:43:46 Now, what happens? This chromosome here: always making beta-gal and it 509 00:43:46 --> 00:43:51 works. This chromosome here: not making beta-gal. 510 00:43:51 --> 00:43:56 Even though it's regulated, it's a mutant. So, in other words, 511 00:43:56 --> 00:44:01 from this very experiment, you can tell that the operator site is only 512 00:44:01 --> 00:44:07 affecting the chromosome that it's physically on, 513 00:44:07 --> 00:44:13 that it doesn't make a protein that floats around. 514 00:44:13 --> 00:44:19 What it does is it's said to work in cys. In cys means on the same 515 00:44:19 --> 00:44:26 chromosome. It physically works on the same chromosome. 516 00:44:26 --> 00:44:32 Now, let's take a look, by contrast, of the properties of 517 00:44:32 --> 00:44:38 the lack repressor mutants. If I give you a lack repressor 518 00:44:38 --> 00:44:43 mutant, I plus, O plus, Z plus is the wild type. 519 00:44:43 --> 00:44:49 I constituative, O plus, Z plus: what happens here? 520 00:44:49 --> 00:44:54 This wild type is one in 1, 00. This guy here: 1,000 and 1, 521 00:44:54 --> 00:45:00 00, and then here let's look at a diploid: 522 00:45:00 --> 00:45:05 I plus, O plus, Z plus, I constituative, 523 00:45:05 --> 00:45:10 O plus, Z plus. What's the effect? The I constituative doesn't make a 524 00:45:10 --> 00:45:15 functioning repressor. But, I plus makes a functioning 525 00:45:15 --> 00:45:21 repressor. So, will this show regulation? 526 00:45:21 --> 00:45:26 Yeah, this will be regulated just fine. This works out just fine, 527 00:45:26 --> 00:45:32 and in fact it'll make 2,000, and it'll make two copies there. 528 00:45:32 --> 00:45:37 But again, the units don't matter too much. And, 529 00:45:37 --> 00:45:42 by contrast, if I give you I plus, O plus, Z minus, and I constituative, 530 00:45:42 --> 00:45:47 O plus, Z plus, what will happen? 531 00:45:47 --> 00:45:53 Here, I have my mutation on this chromosome. But, 532 00:45:53 --> 00:45:58 it doesn't matter because I've got my mutation on this chromosome in 533 00:45:58 --> 00:46:03 the repressor. I've got a mutation on lack Z here, 534 00:46:03 --> 00:46:09 but as long as I have a functional copy, one functional copy of the 535 00:46:09 --> 00:46:15 lack repressor, it works on both chromosomes. 536 00:46:15 --> 00:46:20 It will work on both chromosomes, and so in other words this lack 537 00:46:20 --> 00:46:26 repressor, one copy works on both chromosomes. In other words, 538 00:46:26 --> 00:46:32 it makes a product that diffuses around, and can work on either 539 00:46:32 --> 00:46:38 chromosome, and it's said to work in trans, that is, across. 540 00:46:38 --> 00:46:41 So, the operator is working in cys. It's operating on its own 541 00:46:41 --> 00:46:45 chromosome only. A mutation in the operator only 542 00:46:45 --> 00:46:48 affects the chromosome it lives on, whereas a functional copy of the 543 00:46:48 --> 00:46:52 lack repressor will float around because it's a protein, 544 00:46:52 --> 00:46:55 and that's how Jacobin Maneaux knew the difference. 545 00:46:55 --> 00:46:59 They proved their model by showing that these two kinds of mutations 546 00:46:59 --> 00:47:03 had very different properties. Operator mutations affected only the 547 00:47:03 --> 00:47:07 physical chromosome on which they occurred, which of course they had 548 00:47:07 --> 00:47:11 to infer from the genetics they did, whereas repressor, a functional copy 549 00:47:11 --> 00:47:15 repressor, could act on any chromosome in the cell. 550 00:47:15 --> 00:47:20 So, OK, we've got that. Now, last point, what about glucose? 551 00:47:20 --> 00:47:24 I haven't said a word about glucose. See, this was a big deal to people. 552 00:47:24 --> 00:47:28 This model, the repressor model, we have this repressor. What 553 00:47:28 --> 00:47:36 about glucose? What's glucose doing in this picture? 554 00:47:36 --> 00:47:47 So, glucose control: so here's my gene. Here's my promoter, 555 00:47:47 --> 00:47:58 P lack. Here's my operator, beta-gal. 556 00:47:58 --> 00:48:03 It's encoded by lack Z. You've got all that. When this guy 557 00:48:03 --> 00:48:08 is present, sorry, when lactose is present, 558 00:48:08 --> 00:48:13 the repressor comes off. Polymerase sits down. Wait a second, 559 00:48:13 --> 00:48:18 polymerase isn't supposed to sit down unless there's no glucose. 560 00:48:18 --> 00:48:23 We need another sensor to tell if there's glucose, 561 00:48:23 --> 00:48:28 or if there's low glucose. So, we're going to need us a sensor 562 00:48:28 --> 00:48:34 that tells that. Any ideas? Yep? 563 00:48:34 --> 00:48:40 Yeah, if you work that one through, I don't think it quite works. But, 564 00:48:40 --> 00:48:46 you've got the basic idea. You're going to want another 565 00:48:46 --> 00:48:52 something, and it turns out there's another site over here, 566 00:48:52 --> 00:48:58 OK? There's a second site on which a completely different 567 00:48:58 --> 00:49:05 protein binds. And, this protein is the cyclic AMP 568 00:49:05 --> 00:49:13 regulatory protein, and it so happens that in the cell, 569 00:49:13 --> 00:49:20 when there's low amounts of glucose, let me make sure I've got this right, 570 00:49:20 --> 00:49:28 when there's low amounts of glucose, what we have is high amounts of 571 00:49:28 --> 00:49:34 cyclic AMP. Cyclic AMP turns out, 572 00:49:34 --> 00:49:38 whereas lactose is used directly as the signal, cyclic AMP is used as 573 00:49:38 --> 00:49:42 the signal here. When the cell has low amounts of 574 00:49:42 --> 00:49:46 glucose, it has high amounts of cyclic AMP. Now, 575 00:49:46 --> 00:49:50 what do you want your cyclic AMP to do? How are we going to design this? 576 00:49:50 --> 00:49:54 It's going to bind to a protein, cyclic AMP regulatory protein, it's 577 00:49:54 --> 00:49:58 going to sit down, and now what's it going to do? 578 00:49:58 --> 00:50:02 Is it going to block RNA polymerase? 579 00:50:02 --> 00:50:06 What do we want to do? If there's low glucose, 580 00:50:06 --> 00:50:10 high cyclic AMP, we sit down at the site, we want to turn on 581 00:50:10 --> 00:50:15 transcription now, right? So, what it's got to do is 582 00:50:15 --> 00:50:19 not block RNA polymerase, but help RNA polymerase. So, 583 00:50:19 --> 00:50:24 what it actually does is instead of being a repressor, 584 00:50:24 --> 00:50:28 it's an activator. And what it does is it makes it more attractive for 585 00:50:28 --> 00:50:32 RNA polymerase to bind, and it actually does that by, 586 00:50:32 --> 00:50:36 actually it does it slightly by bending the DNA. 587 00:50:36 --> 00:50:40 But, what it does is it makes it easier for RNA polymerase to bind. 588 00:50:40 --> 00:50:44 It turns out that the promoter is kind of a crummy promoter. 589 00:50:44 --> 00:50:48 It's actually just like, remember the repressor wasn't perfect; the 590 00:50:48 --> 00:50:52 promoter's not perfect either. The promoter's kind of crummy. 591 00:50:52 --> 00:50:56 And, unless RNA polymerase gets a little help from this other 592 00:50:56 --> 00:51:00 regulatory protein, it doesn't work. 593 00:51:00 --> 00:51:04 We have two controls: a negative regulator responding to an 594 00:51:04 --> 00:51:09 environmental cue, a positive activator responding to 595 00:51:09 --> 00:51:13 an environmental cue, helping polymerase decide whether to 596 00:51:13 --> 00:51:18 transcribe or not, and basically that's how a human egg 597 00:51:18 --> 00:51:22 goes to a complete adult and lives its entire life, 598 00:51:22 --> 00:51:27 minus a few other details. There are some details left out, 599 00:51:27 --> 51:32 but that's a sketch of how you turn genes on and off.