1 00:00:16,812 --> 00:00:18,520 BARBARA IMPERIALI: OK, we'll get started. 2 00:00:18,520 --> 00:00:21,020 I've got everything turned on here. 3 00:00:21,020 --> 00:00:22,330 So, a couple of things. 4 00:00:22,330 --> 00:00:26,620 I've been mentioning that it's really kind of cool, 5 00:00:26,620 --> 00:00:28,810 I think, at this stage, where you've 6 00:00:28,810 --> 00:00:31,030 gathered enough steam in some of the topics 7 00:00:31,030 --> 00:00:33,340 that many things that come out in the news 8 00:00:33,340 --> 00:00:36,490 might start looking of interest or relevant to you. 9 00:00:36,490 --> 00:00:38,470 And I found this. 10 00:00:38,470 --> 00:00:41,230 It had a news brief in The Scientist, 11 00:00:41,230 --> 00:00:43,150 and then I went to the original paper, which 12 00:00:43,150 --> 00:00:45,340 is in Nature Biotechnology. 13 00:00:45,340 --> 00:00:49,660 And if you recall, towards the end of the work on proteins, 14 00:00:49,660 --> 00:00:52,550 we were talking about phenylketonuria, 15 00:00:52,550 --> 00:00:56,500 which is a genetically linked disorder, 16 00:00:56,500 --> 00:01:00,400 where people cannot metabolize phenylalanine to keep 17 00:01:00,400 --> 00:01:03,530 the levels of fell phenylalanine in check. 18 00:01:03,530 --> 00:01:05,459 So what happens is the phenylalanine 19 00:01:05,459 --> 00:01:08,350 gets converted to a toxic material, 20 00:01:08,350 --> 00:01:11,560 and it causes a lot of fundamental physiologic 21 00:01:11,560 --> 00:01:17,150 disorders that are a lot of neurologic disorders. 22 00:01:17,150 --> 00:01:20,948 So what a small company in the area called-- 23 00:01:20,948 --> 00:01:22,240 what's the name of the company? 24 00:01:22,240 --> 00:01:23,050 I can see it here. 25 00:01:23,050 --> 00:01:24,610 Anyway. 26 00:01:24,610 --> 00:01:28,900 Synlogic is a synthetic biology company, 27 00:01:28,900 --> 00:01:32,710 which basically engineers bacterial strains 28 00:01:32,710 --> 00:01:36,010 as probiotics that can be used to mitigate 29 00:01:36,010 --> 00:01:37,870 some genetic disorders. 30 00:01:37,870 --> 00:01:41,050 And so they've created a bacterial strain that 31 00:01:41,050 --> 00:01:43,990 can metabolize phenylalanine, so did 32 00:01:43,990 --> 00:01:47,470 a good job of sort of not letting your phenylalanine 33 00:01:47,470 --> 00:01:48,760 levels get too high. 34 00:01:48,760 --> 00:01:53,980 So in the GI system, this bacterium basically 35 00:01:53,980 --> 00:01:56,950 works on phenylalanine to metabolize it 36 00:01:56,950 --> 00:01:59,500 so that, then, people don't have to lead 37 00:01:59,500 --> 00:02:05,980 such a strict dietary regime and have way less risk. 38 00:02:05,980 --> 00:02:07,480 So I think it's a really cool thing. 39 00:02:07,480 --> 00:02:12,400 This probiotic is in one, two clinical trials. 40 00:02:12,400 --> 00:02:14,080 It's being fast tracked. 41 00:02:14,080 --> 00:02:17,290 And the company, in general, is a synthetic biology company 42 00:02:17,290 --> 00:02:22,840 working on solutions to certain types of diseases 43 00:02:22,840 --> 00:02:27,070 that could either save you from having a lot of restrictions 44 00:02:27,070 --> 00:02:29,890 on your lifestyle or, alternatively, save you 45 00:02:29,890 --> 00:02:31,840 from taking medications and stuff. 46 00:02:31,840 --> 00:02:35,320 So that's something that caught my eye. 47 00:02:35,320 --> 00:02:38,440 I want to remind you that I have now put 48 00:02:38,440 --> 00:02:42,310 the link to The Scientist in the sidebar of the website 49 00:02:42,310 --> 00:02:44,740 so it's much easier for you to grab it and take 50 00:02:44,740 --> 00:02:46,330 a look at what's in the news. 51 00:02:46,330 --> 00:02:48,555 There's stuff in the news every two or three days. 52 00:02:48,555 --> 00:02:49,930 And there are things that I think 53 00:02:49,930 --> 00:02:53,860 you'll find interesting that really relate to technology, 54 00:02:53,860 --> 00:02:56,170 engineering, and fundamental science 55 00:02:56,170 --> 00:02:58,360 that are related to biology. 56 00:02:58,360 --> 00:03:01,360 The other thing I want to do is remind you 57 00:03:01,360 --> 00:03:03,910 that towards the end of the class-- 58 00:03:03,910 --> 00:03:06,190 but no time like the present, because it 59 00:03:06,190 --> 00:03:09,410 is sort of the equivalent of one of the problem sets 60 00:03:09,410 --> 00:03:12,610 but, in fact, worth a little bit more than the problem set. 61 00:03:12,610 --> 00:03:17,740 I want to encourage you to keep an eye on The Scientist link 62 00:03:17,740 --> 00:03:20,770 and maybe pick out a topic that two or three of you 63 00:03:20,770 --> 00:03:24,730 would like to write a news brief on with a paragraph of writing 64 00:03:24,730 --> 00:03:27,310 on introduction and what the technology is, 65 00:03:27,310 --> 00:03:31,270 how technology has addressed a particular scientific or 66 00:03:31,270 --> 00:03:33,370 biological problem. 67 00:03:33,370 --> 00:03:36,820 And then there should also be a graphic that describes it. 68 00:03:36,820 --> 00:03:39,910 Not stuff snipped out of whatever you're reading. 69 00:03:39,910 --> 00:03:43,120 Something that you create as a team to sort of 70 00:03:43,120 --> 00:03:45,220 describe a concept to people. 71 00:03:45,220 --> 00:03:49,030 There's alternatives with that assignment. 72 00:03:49,030 --> 00:03:52,700 You can also pick an interesting protein from the PDB, 73 00:03:52,700 --> 00:03:55,480 make a 3D print of it, learn how to print it, 74 00:03:55,480 --> 00:03:58,960 and go to the 3D printers in the maker labs, 75 00:03:58,960 --> 00:04:01,030 and print the protein, and then write 76 00:04:01,030 --> 00:04:02,870 a brief summary of what it is. 77 00:04:02,870 --> 00:04:06,550 And one other idea I had for the engineers' view 78 00:04:06,550 --> 00:04:10,460 is I would love a better, less clunky 79 00:04:10,460 --> 00:04:13,960 topoisomerase demonstration. 80 00:04:13,960 --> 00:04:15,610 In particular, I'd really like one 81 00:04:15,610 --> 00:04:18,550 where you can snip the pieces apart, let 82 00:04:18,550 --> 00:04:21,170 the untangling happen, and put them back together. 83 00:04:21,170 --> 00:04:24,400 So some of you could work with a couple of colleagues 84 00:04:24,400 --> 00:04:26,980 and make something, which I know a lot of you 85 00:04:26,980 --> 00:04:31,450 are really keen on, which is why you're engineers. 86 00:04:31,450 --> 00:04:34,390 OK, any questions about any of this? 87 00:04:34,390 --> 00:04:38,080 I'm trying to make sure that this doesn't creep up on you. 88 00:04:38,080 --> 00:04:40,120 It's just something you do. 89 00:04:40,120 --> 00:04:46,120 It's great to get awareness of technology in the life sciences 90 00:04:46,120 --> 00:04:48,760 because of how much contribution it makes. 91 00:04:48,760 --> 00:04:50,740 So if you keep an eye on things, you 92 00:04:50,740 --> 00:04:53,920 won't be forced to suddenly find something good 93 00:04:53,920 --> 00:04:54,790 at the last minute. 94 00:04:54,790 --> 00:04:56,650 You'll just have found something and go, 95 00:04:56,650 --> 00:04:59,080 that's the perfect thing to describe. 96 00:04:59,080 --> 00:05:00,130 All right? 97 00:05:00,130 --> 00:05:02,300 Questions? 98 00:05:02,300 --> 00:05:05,100 And the other thing is if you're unsure, 99 00:05:05,100 --> 00:05:08,970 you can always let us know what you have chosen 100 00:05:08,970 --> 00:05:12,030 or what you think you're going to choose in the chat with us, 101 00:05:12,030 --> 00:05:15,240 and we'll say, yeah, looks like a good idea. 102 00:05:15,240 --> 00:05:18,490 OK, so let's move forward now. 103 00:05:18,490 --> 00:05:21,310 All right, so what I want to do, first of all, 104 00:05:21,310 --> 00:05:25,510 is just remind you-- it kind of flew by a little bit-- 105 00:05:25,510 --> 00:05:28,954 that DNA replication is bidirectional. 106 00:05:36,830 --> 00:05:38,570 So what that means is wherever you 107 00:05:38,570 --> 00:05:40,910 have an origin of replication, you can 108 00:05:40,910 --> 00:05:43,250 replicate in two directions. 109 00:05:43,250 --> 00:05:46,160 And I was sort of falling asleep thinking about this. 110 00:05:46,160 --> 00:05:48,380 I sure wonder what happens at the other side 111 00:05:48,380 --> 00:05:51,980 of the circular plasmid when the machineries kind of collide 112 00:05:51,980 --> 00:05:54,290 and you spit out a brand-new copy 113 00:05:54,290 --> 00:05:56,240 of a circular chunk of DNA. 114 00:05:56,240 --> 00:05:59,455 But don't think about that too much. 115 00:05:59,455 --> 00:06:01,080 It'll probably keep you awake too much. 116 00:06:01,080 --> 00:06:02,600 So this is circular DNA. 117 00:06:02,600 --> 00:06:06,603 So what organisms have circular DNA? 118 00:06:06,603 --> 00:06:07,520 AUDIENCE: Prokaryotes. 119 00:06:07,520 --> 00:06:08,812 BARBARA IMPERIALI: Prokaryotes. 120 00:06:08,812 --> 00:06:11,135 Remember, the eukaryotic DNA is linear. 121 00:06:11,135 --> 00:06:12,510 And actually, we're going to talk 122 00:06:12,510 --> 00:06:16,170 about a conundrum with the eukaryotic DNA because 123 00:06:16,170 --> 00:06:19,780 of the ends of the DNA, the ends of the chromosomes 124 00:06:19,780 --> 00:06:22,650 and their copying, when we talk about telomerase. 125 00:06:22,650 --> 00:06:25,590 But this is typical of a bacterial circular DNA. 126 00:06:25,590 --> 00:06:29,250 It's usually super coiled and becomes uncoiled in order 127 00:06:29,250 --> 00:06:30,600 to be replicated. 128 00:06:30,600 --> 00:06:33,030 So, obviously, going bidirectionally 129 00:06:33,030 --> 00:06:35,490 gives you twice the speed, because your roaring 130 00:06:35,490 --> 00:06:37,350 around the same time. 131 00:06:37,350 --> 00:06:40,530 The helicase opens up the DNA. 132 00:06:40,530 --> 00:06:42,720 DNA polymerase does its job. 133 00:06:42,720 --> 00:06:48,340 The pink strand here would be the leading strand. 134 00:06:48,340 --> 00:06:53,170 And there's obviously going to be one on both strands of DNA, 135 00:06:53,170 --> 00:06:55,067 which will replicate very well. 136 00:06:55,067 --> 00:06:56,650 But don't forget, you're going to have 137 00:06:56,650 --> 00:07:00,620 to deal with the lagging strands in both cases. 138 00:07:00,620 --> 00:07:09,330 So in both cases, what you would do 139 00:07:09,330 --> 00:07:13,500 is stick down a primer in order to be 140 00:07:13,500 --> 00:07:16,110 able to build those lagging strands. 141 00:07:16,110 --> 00:07:18,930 Otherwise, DNA polymerase can't get a grip 142 00:07:18,930 --> 00:07:21,360 on the double-stranded DNA in order 143 00:07:21,360 --> 00:07:23,220 to carry out the synthesis. 144 00:07:23,220 --> 00:07:26,730 So in those cases, there would be a primer 145 00:07:26,730 --> 00:07:30,390 to set up the lagging strand, and then DNA synthesis 146 00:07:30,390 --> 00:07:31,720 can occur. 147 00:07:31,720 --> 00:07:32,910 So let's put that in. 148 00:07:36,840 --> 00:07:47,280 Once there's a primer here, DNA synthesis can occur. 149 00:07:47,280 --> 00:07:50,330 And then what happens with respect to the pieces 150 00:07:50,330 --> 00:07:51,680 that we're dealing with? 151 00:07:51,680 --> 00:07:55,380 What happens with the primer? 152 00:07:55,380 --> 00:07:59,070 What do we have to do to get to a nice complete, intact strand 153 00:07:59,070 --> 00:07:59,910 of DNA? 154 00:07:59,910 --> 00:08:02,620 Which enzymes are involved? 155 00:08:02,620 --> 00:08:03,314 Yeah? 156 00:08:03,314 --> 00:08:04,022 AUDIENCE: Ligase. 157 00:08:06,617 --> 00:08:08,950 BARBARA IMPERIALI: Here, there would be ligase activity. 158 00:08:08,950 --> 00:08:09,803 We need that. 159 00:08:09,803 --> 00:08:10,970 What about at the other end? 160 00:08:10,970 --> 00:08:13,300 What do we do with the primer, and then how 161 00:08:13,300 --> 00:08:15,600 can we move forward? 162 00:08:15,600 --> 00:08:17,620 Someone else? 163 00:08:17,620 --> 00:08:19,090 What happens with the primer? 164 00:08:19,090 --> 00:08:21,220 It's oftentimes an RNA primer. 165 00:08:21,220 --> 00:08:24,490 You'll see in a moment that RNA doesn't need a primase, 166 00:08:24,490 --> 00:08:27,250 so it's very easy for RNA polymerase 167 00:08:27,250 --> 00:08:28,690 to stitch in little pieces. 168 00:08:28,690 --> 00:08:34,929 What do we have to do, though, to get an intact strand of DNA? 169 00:08:34,929 --> 00:08:35,634 OK. 170 00:08:35,634 --> 00:08:37,451 AUDIENCE: Have to remove the [? RNA. ?] 171 00:08:37,451 --> 00:08:38,409 BARBARA IMPERIALI: Yup. 172 00:08:38,409 --> 00:08:41,200 So you're going to remove the RNA. 173 00:08:41,200 --> 00:08:44,830 Then the polymerase, later on, when we keep going, 174 00:08:44,830 --> 00:08:46,990 can sort of build this piece. 175 00:08:46,990 --> 00:08:49,630 And then we'll have to ligate that, as well. 176 00:08:49,630 --> 00:08:54,490 So you want to remember all the functions of those enzymes that 177 00:08:54,490 --> 00:08:56,570 are involved in replication. 178 00:08:56,570 --> 00:08:58,510 It's a little worrisome that people-- 179 00:08:58,510 --> 00:09:00,220 I know you don't want to talk or you 180 00:09:00,220 --> 00:09:02,860 think that's an obvious answer, but it's really important 181 00:09:02,860 --> 00:09:04,990 that you have them at your fingertips, some 182 00:09:04,990 --> 00:09:07,660 of these enzymes that are involved in this process. 183 00:09:07,660 --> 00:09:09,760 Because they should start to become second nature. 184 00:09:09,760 --> 00:09:15,070 When you have to make a full DNA copy of an entire genome, 185 00:09:15,070 --> 00:09:17,180 there's a lot of moving parts. 186 00:09:17,180 --> 00:09:20,230 But if you start walking through the logic of them, 187 00:09:20,230 --> 00:09:21,640 they make sense. 188 00:09:21,640 --> 00:09:24,370 If I'm going to unpeel DNA, I need a helicase. 189 00:09:24,370 --> 00:09:26,530 If I'm going to keep it single stranded, 190 00:09:26,530 --> 00:09:29,230 I need single strand binding proteins. 191 00:09:29,230 --> 00:09:32,470 If I'm going to move forward, sure, I need the polymerase, 192 00:09:32,470 --> 00:09:36,250 but what does the polymerase need? 193 00:09:36,250 --> 00:09:39,220 It needs a primer in order to have double strand, 194 00:09:39,220 --> 00:09:41,260 because DNA polymerase only wants 195 00:09:41,260 --> 00:09:46,000 to lock onto a double strand to go start doing its job. 196 00:09:46,000 --> 00:09:47,860 These complications with the lagging 197 00:09:47,860 --> 00:09:50,050 strands that are really annoying, 198 00:09:50,050 --> 00:09:53,320 but it's pretty remarkable nature has addressed this 199 00:09:53,320 --> 00:09:56,890 and is able, remember, to replicate DNA 200 00:09:56,890 --> 00:10:03,560 and bacteria at a speed of 100 base pairs per second. 201 00:10:03,560 --> 00:10:06,470 So that's what's going on, this entire process. 202 00:10:06,470 --> 00:10:06,970 Excuse me. 203 00:10:06,970 --> 00:10:09,160 1,000 base pairs a second. 204 00:10:09,160 --> 00:10:11,140 I just want you to remember this process is 205 00:10:11,140 --> 00:10:13,390 slower in eukaryotes. 206 00:10:13,390 --> 00:10:18,610 It's about 30 to 50 base pairs per second. 207 00:10:18,610 --> 00:10:21,070 Obviously, when you're speeding, you make more mistakes, 208 00:10:21,070 --> 00:10:28,390 so there are more mistakes in bacterial genome replication. 209 00:10:28,390 --> 00:10:31,390 Why does it not matter so much if there's 210 00:10:31,390 --> 00:10:35,380 a mistake in a bacterial genome? 211 00:10:35,380 --> 00:10:37,960 What do you know about bacteria and their lifestyles? 212 00:10:37,960 --> 00:10:39,820 Do they stick around a long time? 213 00:10:39,820 --> 00:10:40,320 No. 214 00:10:40,320 --> 00:10:42,340 So they divide quickly. 215 00:10:42,340 --> 00:10:43,730 They live and die quickly. 216 00:10:43,730 --> 00:10:48,610 So you're not having to keep an intact genome without mistakes 217 00:10:48,610 --> 00:10:51,700 in it for a long time, because you're just 218 00:10:51,700 --> 00:10:52,690 turning over bacteria. 219 00:10:52,690 --> 00:10:55,210 If there's a mistake, it probably dies out. 220 00:10:55,210 --> 00:10:59,560 Or heaven forbid, there's resistance to drugs developed. 221 00:10:59,560 --> 00:11:01,060 And we'll talk about those later, 222 00:11:01,060 --> 00:11:03,460 because those occur due to mistakes 223 00:11:03,460 --> 00:11:05,740 and in bacterial replication. 224 00:11:05,740 --> 00:11:09,610 But in a eukaryotic genome, we have 225 00:11:09,610 --> 00:11:12,310 to preserve the integrity of the genome. 226 00:11:12,310 --> 00:11:15,910 So I'm going to talk about two things that 227 00:11:15,910 --> 00:11:18,920 are related to the accuracy of replication 228 00:11:18,920 --> 00:11:21,520 now, because that's a really important component. 229 00:11:21,520 --> 00:11:27,670 All right, so the first thing is to think about, 230 00:11:27,670 --> 00:11:31,542 what's the basal rate of making mistakes of DNA polymerase? 231 00:11:31,542 --> 00:11:33,000 So for that purpose, I'm just going 232 00:11:33,000 --> 00:11:41,700 to put down a piece of DNA with its partner that's 233 00:11:41,700 --> 00:11:46,410 being synthesized, five prime to three prime. 234 00:11:46,410 --> 00:11:50,370 And I'll put in some bases, so A. So that 235 00:11:50,370 --> 00:11:52,440 would have had a T put in opposite. 236 00:11:55,210 --> 00:11:56,800 G, that would have had a C. 237 00:11:56,800 --> 00:11:59,320 So these have two hydrogen bonds. 238 00:11:59,320 --> 00:12:01,510 These have three hydrogen bonds. 239 00:12:01,510 --> 00:12:04,870 And let's say we now have a C here. 240 00:12:04,870 --> 00:12:08,080 So we want to put in a G at the position opposite. 241 00:12:08,080 --> 00:12:13,150 DNA polymerase wants to add the next base pair. 242 00:12:13,150 --> 00:12:15,730 It should be a G, because it's going 243 00:12:15,730 --> 00:12:19,660 in opposite a C. It's being grown in the right direction, 244 00:12:19,660 --> 00:12:21,370 five prime to three prime. 245 00:12:21,370 --> 00:12:26,560 So the basal error rate is about 1,000 to 1. 246 00:12:26,560 --> 00:12:32,920 So 999 times out of 1,000, the right base gets put in. 247 00:12:32,920 --> 00:12:36,280 1 time out of 1,000, you might put in the wrong rate. 248 00:12:36,280 --> 00:12:49,220 So the error rate is about 1 in 10 to the 3. 249 00:12:49,220 --> 00:12:50,720 That's really all that's-- 250 00:12:50,720 --> 00:12:53,480 all that's at play here is energetics, 251 00:12:53,480 --> 00:12:57,050 just how favorable putting in the right base is. 252 00:12:57,050 --> 00:13:00,860 But there's a slight chance that the wrong base will just go in. 253 00:13:00,860 --> 00:13:05,203 Because the energetics are sufficiently different, 254 00:13:05,203 --> 00:13:06,620 but you're going to make mistakes, 255 00:13:06,620 --> 00:13:09,500 just because of the thermodynamic balance. 256 00:13:09,500 --> 00:13:12,070 If you're putting in 999, you're going 257 00:13:12,070 --> 00:13:15,380 to get it wrong some of the time, just statistically, 258 00:13:15,380 --> 00:13:18,290 because the difference in energy between putting 259 00:13:18,290 --> 00:13:21,290 in the right base or putting in a wrong base. 260 00:13:21,290 --> 00:13:23,840 So that error rate is too high. 261 00:13:23,840 --> 00:13:27,860 If we replicated our genome, 32 billion base pairs, 262 00:13:27,860 --> 00:13:31,430 and we had a 1 in 10 to the 3 error rate, 263 00:13:31,430 --> 00:13:34,460 we'd have a lot of mistakes in the genome, right? 264 00:13:34,460 --> 00:13:37,820 And we cannot tolerate that, because all those mistakes 265 00:13:37,820 --> 00:13:41,630 in our genome will then propagate to mistakes 266 00:13:41,630 --> 00:13:45,330 in our proteome, if we're in the right segments of the genome. 267 00:13:45,330 --> 00:13:48,620 So this is pretty unacceptable with respect to an error rate. 268 00:13:54,430 --> 00:13:57,550 So one way that nature deals with this 269 00:13:57,550 --> 00:14:05,850 is that DNA polymerase actually does some proofreading, 270 00:14:05,850 --> 00:14:07,290 all right? 271 00:14:07,290 --> 00:14:09,315 So it has a proofreading function. 272 00:14:15,670 --> 00:14:16,420 So what do you do? 273 00:14:16,420 --> 00:14:18,400 When you're proofreading, you take a quick look 274 00:14:18,400 --> 00:14:21,025 at what you've just written and say, oh, yeah, that looks good. 275 00:14:21,025 --> 00:14:21,940 That looks good. 276 00:14:21,940 --> 00:14:24,190 So what DNA polymerase is-- it more or less 277 00:14:24,190 --> 00:14:29,110 reaches back to the base it puts in and checks that it's OK. 278 00:14:29,110 --> 00:14:31,600 It can only proofread one base back. 279 00:14:31,600 --> 00:14:34,420 It can't proofread from work that's 280 00:14:34,420 --> 00:14:35,830 been done a long time ago. 281 00:14:35,830 --> 00:14:38,920 It can just proofread very recent work. 282 00:14:38,920 --> 00:14:41,590 And if it looks like it's the wrong base, 283 00:14:41,590 --> 00:14:44,730 DNA polymerase has an opposite function. 284 00:14:44,730 --> 00:14:49,655 It has what's known as a three prime exonuclease activity. 285 00:14:49,655 --> 00:14:51,280 So I'll write that down, and then we'll 286 00:14:51,280 --> 00:14:52,900 talk about what that means. 287 00:14:52,900 --> 00:14:56,020 Three prime exonuclease. 288 00:15:01,030 --> 00:15:07,390 So let's say we put in, instead of G, we put in a T. 289 00:15:07,390 --> 00:15:08,590 That's bad news. 290 00:15:08,590 --> 00:15:12,625 So what it can do is it can reach back and cut off, 291 00:15:12,625 --> 00:15:19,180 from the three prime end, a single nucleotide, the one that 292 00:15:19,180 --> 00:15:20,890 just got put in, all right? 293 00:15:20,890 --> 00:15:23,950 And then allow the process to reoccur 294 00:15:23,950 --> 00:15:26,210 to get the right base pair in. 295 00:15:26,210 --> 00:15:28,840 So a lot of enzymes will catalyze both forwards 296 00:15:28,840 --> 00:15:30,790 and backwards reactions. 297 00:15:30,790 --> 00:15:33,790 DNA polymerase, the energetics of such 298 00:15:33,790 --> 00:15:36,880 are that it is able to catalyze both 299 00:15:36,880 --> 00:15:40,270 the addition of a nucleotide and the removal 300 00:15:40,270 --> 00:15:44,710 of a single nucleotide, only if it's at the three prime end. 301 00:15:44,710 --> 00:15:48,910 Only if it's at an open end, where it's just been put in. 302 00:15:48,910 --> 00:15:51,730 So, remember, the DNA polymerase is still here, 303 00:15:51,730 --> 00:15:54,640 because its plan is to move forward and keep 304 00:15:54,640 --> 00:15:56,560 on putting in nucleotides. 305 00:15:56,560 --> 00:15:58,330 But it actually checks back. 306 00:15:58,330 --> 00:16:00,130 You could picture DNA pol just sort 307 00:16:00,130 --> 00:16:02,050 of quickly looking over its shoulder 308 00:16:02,050 --> 00:16:05,500 at the work it's just done and realizing that's the wrong one. 309 00:16:05,500 --> 00:16:10,720 So what this does is brings the error rates down considerably. 310 00:16:16,350 --> 00:16:19,950 Goes in 1 in 10 to the 5. 311 00:16:19,950 --> 00:16:21,330 So that's way better. 312 00:16:21,330 --> 00:16:25,630 1 in 100,000 is much better, and that's pretty acceptable. 313 00:16:25,630 --> 00:16:29,100 So it means you're really making very minimal mistakes 314 00:16:29,100 --> 00:16:30,990 in the replication. 315 00:16:30,990 --> 00:16:36,800 So this part, the proofreading, brings the error rate 316 00:16:36,800 --> 00:16:40,790 from 1 in 10 to the 3 to 1 in 10 to the 5. 317 00:16:40,790 --> 00:16:46,670 But it can only work during DNA polymerase activity, 318 00:16:46,670 --> 00:16:51,200 and it can only work on the most recent nucleotide that 319 00:16:51,200 --> 00:16:52,910 has been put in. 320 00:16:52,910 --> 00:16:56,176 So this is basically a summary of-- yes, question? 321 00:16:56,176 --> 00:16:59,060 AUDIENCE: So are [INAUDIBLE] in prokaryotes and eukaryotes? 322 00:16:59,060 --> 00:17:02,240 BARBARA IMPERIALI: They are, and they are similar-- 323 00:17:02,240 --> 00:17:06,500 actually, there's slightly less error rates in eukaryotes, 324 00:17:06,500 --> 00:17:09,060 because the speed is slower. 325 00:17:09,060 --> 00:17:11,079 So the opportunity to fix things is going 326 00:17:11,079 --> 00:17:12,750 to be a little bit better. 327 00:17:12,750 --> 00:17:15,140 So, in the end, your goal, really, 328 00:17:15,140 --> 00:17:18,680 is to bring your error rate between 10 to the 5 and 10 329 00:17:18,680 --> 00:17:19,790 to the 6. 330 00:17:19,790 --> 00:17:23,839 But for bacteria, because the speed is so much larger, 331 00:17:23,839 --> 00:17:26,480 this is sort of the limit of it. 332 00:17:26,480 --> 00:17:29,450 But in eukaryote, it can be a little bit better, 333 00:17:29,450 --> 00:17:31,220 because the speed is slower. 334 00:17:31,220 --> 00:17:33,980 So you could imagine, if you're quickly proofreading, 335 00:17:33,980 --> 00:17:36,560 you're doing a less good job than if you're slowly 336 00:17:36,560 --> 00:17:37,700 proofreading. 337 00:17:37,700 --> 00:17:39,890 But the enzymes that I'll talk to you 338 00:17:39,890 --> 00:17:44,930 about in a second in eukarya are to fix entire work that's 339 00:17:44,930 --> 00:17:45,950 already been done. 340 00:17:45,950 --> 00:17:47,370 We'll see that in a moment. 341 00:17:47,370 --> 00:17:48,980 And that's what really cleans up. 342 00:17:48,980 --> 00:17:53,000 These enzymes are called the guardians of the genome, 343 00:17:53,000 --> 00:17:56,120 and they are much more sophisticated in eukarya. 344 00:17:56,120 --> 00:17:57,680 So that's a very good point. 345 00:17:57,680 --> 00:18:01,460 All right, so here's the general scheme. 346 00:18:01,460 --> 00:18:02,760 There is an extension. 347 00:18:02,760 --> 00:18:04,070 There's a mistake. 348 00:18:04,070 --> 00:18:05,390 So there's proofreading. 349 00:18:05,390 --> 00:18:07,040 The mistake gets taken out. 350 00:18:07,040 --> 00:18:10,940 And then you keep on extending again, all right? 351 00:18:10,940 --> 00:18:15,900 So this now becomes pretty good. 352 00:18:15,900 --> 00:18:18,080 But what we need to talk about now is, 353 00:18:18,080 --> 00:18:21,050 what are the enzymes that go to work-- 354 00:18:21,050 --> 00:18:22,730 and I'm leaving what's on that board 355 00:18:22,730 --> 00:18:24,605 there, because I'm going to come back to it-- 356 00:18:26,900 --> 00:18:31,990 to actually correct mistakes in DNA. 357 00:18:31,990 --> 00:18:40,120 So let's talk about the guardians of the genome. 358 00:18:44,340 --> 00:18:48,930 Because remember that your DNA is your permanent record 359 00:18:48,930 --> 00:18:50,850 of what needs to be made. 360 00:18:50,850 --> 00:18:54,000 All right, so the types of mistakes 361 00:18:54,000 --> 00:18:55,650 that can be fixed with proofreading 362 00:18:55,650 --> 00:18:57,225 are only recent mistakes. 363 00:18:57,225 --> 00:18:59,100 The types of enzymes I'm going to talk to you 364 00:18:59,100 --> 00:19:02,010 about mistakes that are found globally 365 00:19:02,010 --> 00:19:03,735 within double-stranded DNA. 366 00:19:13,650 --> 00:19:15,300 And these are mistakes wherever. 367 00:19:15,300 --> 00:19:19,230 They may be mistakes that didn't get corrected by proofreading. 368 00:19:19,230 --> 00:19:21,150 But more importantly, they're mistakes 369 00:19:21,150 --> 00:19:24,960 that occur due to some kind of damage on the DNA. 370 00:19:24,960 --> 00:19:29,790 So what kind of things might impact the integrity of our DNA 371 00:19:29,790 --> 00:19:32,400 on a day-to-day basis? 372 00:19:32,400 --> 00:19:33,160 Yeah? 373 00:19:33,160 --> 00:19:33,630 AUDIENCE: Sunlight. 374 00:19:33,630 --> 00:19:34,797 BARBARA IMPERIALI: Sunlight. 375 00:19:34,797 --> 00:19:37,170 So terrible stuff. 376 00:19:37,170 --> 00:19:40,340 UV light will actually cause some cross-links, 377 00:19:40,340 --> 00:19:43,590 and I'll show you one of those, that are very serious in DNA. 378 00:19:43,590 --> 00:19:47,070 What else might hurt the genome? 379 00:19:47,070 --> 00:19:48,300 So sunlight. 380 00:19:48,300 --> 00:19:50,168 People say, don't go out in the sunlight. 381 00:19:50,168 --> 00:19:51,210 They're right about that. 382 00:19:51,210 --> 00:19:52,500 What else? 383 00:19:52,500 --> 00:19:53,073 Yeah? 384 00:19:53,073 --> 00:19:53,935 AUDIENCE: Radiation. 385 00:19:53,935 --> 00:19:55,810 BARBARA IMPERIALI: Radiation is another form. 386 00:19:55,810 --> 00:19:58,500 So that's like radioactivity. 387 00:19:58,500 --> 00:20:00,630 Radiation is important, right? 388 00:20:00,630 --> 00:20:03,930 So if our ozone layer gets thin, there's more risk there, 389 00:20:03,930 --> 00:20:05,040 as well. 390 00:20:05,040 --> 00:20:06,030 What about barbecue? 391 00:20:06,030 --> 00:20:06,563 Yeah? 392 00:20:06,563 --> 00:20:07,730 AUDIENCE: Harmful chemicals. 393 00:20:07,730 --> 00:20:08,730 BARBARA IMPERIALI: Yeah. 394 00:20:08,730 --> 00:20:11,340 Awful chemicals, terrible chemicals, right. 395 00:20:11,340 --> 00:20:14,610 So chemicals. 396 00:20:14,610 --> 00:20:19,290 So these are things like polyaromatic structures 397 00:20:19,290 --> 00:20:22,590 that actually slip into your genome and cause mistakes 398 00:20:22,590 --> 00:20:23,730 in the reading. 399 00:20:23,730 --> 00:20:26,130 Or they actually physically modify 400 00:20:26,130 --> 00:20:29,340 the bases to make it a base that doesn't look like a base 401 00:20:29,340 --> 00:20:30,130 anymore. 402 00:20:30,130 --> 00:20:34,340 So these are commonly very reactive chemicals. 403 00:20:34,340 --> 00:20:37,880 So these are all very serious things to the genome. 404 00:20:37,880 --> 00:20:42,250 And so the enzymes that mitigate the damage to the DNA 405 00:20:42,250 --> 00:20:45,940 basically screen along the double-stranded DNA 406 00:20:45,940 --> 00:20:47,980 to look for defects. 407 00:20:47,980 --> 00:20:51,940 Because if you have perfectly paired DNA, then you're not-- 408 00:20:51,940 --> 00:20:55,330 you're going to have a very regular structure. 409 00:20:55,330 --> 00:20:57,785 Whereas, if something has happened to the DNA, 410 00:20:57,785 --> 00:20:59,410 there's something wrong with the base-- 411 00:20:59,410 --> 00:21:03,580 it's not base pairing well or something has actually 412 00:21:03,580 --> 00:21:05,350 happened between bases where they're 413 00:21:05,350 --> 00:21:07,360 causing a bulge in the genome-- 414 00:21:07,360 --> 00:21:10,000 then these enzymes will come into play. 415 00:21:10,000 --> 00:21:12,520 About a few years ago-- 416 00:21:12,520 --> 00:21:17,340 actually, it was on a class day, so I always enjoy these. 417 00:21:17,340 --> 00:21:21,310 The Nobel Prize was awarded in 2015 418 00:21:21,310 --> 00:21:26,290 for the researchers who deciphered the mechanisms 419 00:21:26,290 --> 00:21:30,200 for correcting the genome through DNA repair mechanisms. 420 00:21:30,200 --> 00:21:32,443 So there are two basic mechanisms 421 00:21:32,443 --> 00:21:33,610 that I'll talk to you about. 422 00:21:38,140 --> 00:21:43,215 One is base excision repair. 423 00:21:49,650 --> 00:21:53,200 And the other one is a lot more serious. 424 00:21:53,200 --> 00:21:56,170 It's actually the entire nucleotide excision repair. 425 00:21:56,170 --> 00:21:59,430 So one fixes just the base that's gone wrong, 426 00:21:59,430 --> 00:22:02,700 but the other one takes much more of the structure out 427 00:22:02,700 --> 00:22:03,870 to fix it. 428 00:22:03,870 --> 00:22:12,320 So it's nucleotide excision repair. 429 00:22:12,320 --> 00:22:18,250 So BER and NER, and we're going to talk 430 00:22:18,250 --> 00:22:21,340 about both of those mechanisms, because they're 431 00:22:21,340 --> 00:22:22,390 very fascinating. 432 00:22:22,390 --> 00:22:25,450 And they kind of lean on some of what you've learned already. 433 00:22:25,450 --> 00:22:28,660 So base excision repair occurs when 434 00:22:28,660 --> 00:22:30,430 there's a defect in the base. 435 00:22:30,430 --> 00:22:31,810 Maybe it's the wrong base. 436 00:22:31,810 --> 00:22:34,580 Maybe it's just been modified a little bit. 437 00:22:34,580 --> 00:22:38,635 So what happens in base excision repair is that the base gets-- 438 00:22:38,635 --> 00:22:40,510 all right, I'm going to just use the pointer, 439 00:22:40,510 --> 00:22:42,730 because my little spotlight-- it'll pop back. 440 00:22:42,730 --> 00:22:43,960 It's a bit magical. 441 00:22:43,960 --> 00:22:48,070 So base excision repair, only the base, a single localized 442 00:22:48,070 --> 00:22:49,660 base, is damaged. 443 00:22:49,660 --> 00:22:52,640 There is some chemistry, for example, 444 00:22:52,640 --> 00:22:57,460 that will convert cytosine to uracil called a deamination 445 00:22:57,460 --> 00:22:58,630 mechanism. 446 00:22:58,630 --> 00:23:02,320 And in fact, if you replace the cytosine with a uracil, 447 00:23:02,320 --> 00:23:04,090 then you get in trouble with respect 448 00:23:04,090 --> 00:23:08,830 to its base pairing with its appropriate purine partner. 449 00:23:08,830 --> 00:23:12,160 So in base excision repair, what will happen 450 00:23:12,160 --> 00:23:14,770 is that base will be detected. 451 00:23:14,770 --> 00:23:19,660 It will be flipped out from the context of double-stranded DNA. 452 00:23:19,660 --> 00:23:22,180 If it's tucked in the double-stranded DNA, 453 00:23:22,180 --> 00:23:24,340 you can't quite cut the bond that's 454 00:23:24,340 --> 00:23:27,130 attached from the ribose sugar to the base. 455 00:23:27,130 --> 00:23:30,490 So the base gets flipped out of the DNA structure. 456 00:23:30,490 --> 00:23:33,400 And there's an enzyme known as a glycosylase that 457 00:23:33,400 --> 00:23:36,970 cuts the bond between the ribose and the base 458 00:23:36,970 --> 00:23:38,990 and gets rid of it. 459 00:23:38,990 --> 00:23:42,520 And then what happens is the rest of the nucleotide, 460 00:23:42,520 --> 00:23:45,490 just that one nucleotide, gets removed. 461 00:23:45,490 --> 00:23:48,520 And then DNA polymerase fills the gap, 462 00:23:48,520 --> 00:23:50,980 and the strand is sealed by a ligase. 463 00:23:50,980 --> 00:23:55,300 So a glycosylase cuts the base out. 464 00:23:55,300 --> 00:23:57,460 There's a couple of enzymes that actually 465 00:23:57,460 --> 00:24:02,470 cut the ribose, the phosphodiester linkages out. 466 00:24:02,470 --> 00:24:04,720 And then the two enzymes, remember, 467 00:24:04,720 --> 00:24:06,610 that are important when we're making 468 00:24:06,610 --> 00:24:11,500 DNA, the polymerase and the ligase, 469 00:24:11,500 --> 00:24:14,920 work together to put a base into this position. 470 00:24:17,430 --> 00:24:23,560 And then they put the base back in, 471 00:24:23,560 --> 00:24:25,390 and then the ligase joins the gap. 472 00:24:25,390 --> 00:24:28,420 So DNA polymerase will make one of the bonds. 473 00:24:28,420 --> 00:24:30,970 The ligase will make the other bond, all right? 474 00:24:30,970 --> 00:24:36,730 So that's base excision repair, and it's 475 00:24:36,730 --> 00:24:40,870 monitored by finding that there is a lack of integrity 476 00:24:40,870 --> 00:24:43,060 in the double-stranded DNA. 477 00:24:43,060 --> 00:24:45,220 It's only a base that's affected, 478 00:24:45,220 --> 00:24:47,380 so that base gets removed. 479 00:24:47,380 --> 00:24:50,560 The rest of the nucleotide is removed, but only one of them. 480 00:24:50,560 --> 00:24:53,140 And then they're replaced through the concerted action 481 00:24:53,140 --> 00:24:55,330 of polymerase and ligase. 482 00:24:55,330 --> 00:24:58,900 Now, there is another mechanism that's much more serious, 483 00:24:58,900 --> 00:25:01,810 and this is very typical of the types of damage 484 00:25:01,810 --> 00:25:05,620 that get formed from sunlight and UV radiation, 485 00:25:05,620 --> 00:25:09,340 is when two thymidines are adjacent to each other, they 486 00:25:09,340 --> 00:25:13,420 will undergo, quite commonly, a chemical reaction where 487 00:25:13,420 --> 00:25:15,610 they form a dimeric structure. 488 00:25:15,610 --> 00:25:19,930 So there's much more wrong with the DNA 489 00:25:19,930 --> 00:25:21,890 strand in that situation. 490 00:25:21,890 --> 00:25:23,770 So those get noticed. 491 00:25:23,770 --> 00:25:26,380 This thing's driving me nuts. 492 00:25:26,380 --> 00:25:30,370 Those get noticed as a real defect in the DNA. 493 00:25:30,370 --> 00:25:32,720 Base excision repair won't work. 494 00:25:32,720 --> 00:25:33,610 Why wouldn't it work? 495 00:25:36,140 --> 00:25:36,685 Yeah? 496 00:25:36,685 --> 00:25:38,560 AUDIENCE: Because the two are bound together. 497 00:25:38,560 --> 00:25:39,640 BARBARA IMPERIALI: Yeah, they're bound together. 498 00:25:39,640 --> 00:25:41,200 You can't peel them back out. 499 00:25:41,200 --> 00:25:43,030 You can't break in that structure. 500 00:25:43,030 --> 00:25:48,300 So you've got to move to a much more sort of major fixing 501 00:25:48,300 --> 00:25:52,250 of the DNA strand. 502 00:25:52,250 --> 00:25:55,000 So there is a genetically linked disorder 503 00:25:55,000 --> 00:25:59,230 where the enzymes involved in nucleotide excision repair 504 00:25:59,230 --> 00:26:00,640 do not work. 505 00:26:00,640 --> 00:26:05,200 And when a child has a copy of both defective genes 506 00:26:05,200 --> 00:26:08,860 from the parents, both one from the mother and the father, 507 00:26:08,860 --> 00:26:12,820 it's impossible for them to fix these defects in the DNA. 508 00:26:12,820 --> 00:26:15,940 And they get a lot of physiologic problems 509 00:26:15,940 --> 00:26:21,070 like sort of scarring and sunburns, barely anything 510 00:26:21,070 --> 00:26:22,110 at all. 511 00:26:22,110 --> 00:26:27,940 So this is a group of children that are so afflicted with this 512 00:26:27,940 --> 00:26:31,150 genetic disorders that they cannot go out in the daytime 513 00:26:31,150 --> 00:26:32,080 at all. 514 00:26:32,080 --> 00:26:35,200 So you'll sometimes see they're called Children of the Night, 515 00:26:35,200 --> 00:26:37,840 because basically, they have to flip their schedules. 516 00:26:37,840 --> 00:26:39,843 They just can't go near sunlight. 517 00:26:39,843 --> 00:26:41,260 And if they go out, they basically 518 00:26:41,260 --> 00:26:43,180 have to be covered from head to toe. 519 00:26:43,180 --> 00:26:45,820 And that's including their eyes, because you 520 00:26:45,820 --> 00:26:47,770 can get sunburn of the eyes. 521 00:26:47,770 --> 00:26:50,590 You can sort of see, in some of these pictures, 522 00:26:50,590 --> 00:26:53,860 that it looks really serious defects. 523 00:26:53,860 --> 00:26:57,490 And this is just the external manifestation. 524 00:26:57,490 --> 00:27:02,120 The internal manifestation would be cases of skin cancer very, 525 00:27:02,120 --> 00:27:03,290 very readily. 526 00:27:03,290 --> 00:27:06,460 So if you don't have at least one good copy 527 00:27:06,460 --> 00:27:09,520 of the enzyme that does nucleotide excision repair, 528 00:27:09,520 --> 00:27:11,020 then you're in trouble. 529 00:27:11,020 --> 00:27:13,030 And the defects are called-- 530 00:27:13,030 --> 00:27:16,070 it's called xeroderma pigmentosum. 531 00:27:16,070 --> 00:27:18,130 And there's actually a lot of family groups 532 00:27:18,130 --> 00:27:21,160 that get together, because the best way is just 533 00:27:21,160 --> 00:27:25,060 to form a sort of social network so the children understand 534 00:27:25,060 --> 00:27:28,120 what each other-- the limitations that they all have. 535 00:27:28,120 --> 00:27:30,010 And they can play together and be 536 00:27:30,010 --> 00:27:32,020 on these sort of flipped schedules 537 00:27:32,020 --> 00:27:35,230 in order to avoid any sunlight. 538 00:27:35,230 --> 00:27:39,730 So, in this case, it's essential to basically clip out 539 00:27:39,730 --> 00:27:41,830 a large chunk of the DNA. 540 00:27:41,830 --> 00:27:46,450 So what happens in this case is that the DNA is recognized, 541 00:27:46,450 --> 00:27:49,960 and then a large portion of it-- about a dozen nucleotides-- 542 00:27:49,960 --> 00:27:51,250 are cleaved out. 543 00:27:51,250 --> 00:27:55,630 And then, once again, DNA polymerase fills the gap, 544 00:27:55,630 --> 00:27:58,630 and DNA ligase seals the last gap. 545 00:27:58,630 --> 00:28:01,660 So DNA polymerase will be able to fill 546 00:28:01,660 --> 00:28:04,090 going from the five prime to three prime. 547 00:28:04,090 --> 00:28:07,090 There'll still be one gap, and then the ligase fixes it. 548 00:28:07,090 --> 00:28:09,190 So I think these kinds of things that 549 00:28:09,190 --> 00:28:11,590 are done to mitigate damage on the genome 550 00:28:11,590 --> 00:28:13,750 are very important to understand, 551 00:28:13,750 --> 00:28:16,090 because this is happening all the time. 552 00:28:16,090 --> 00:28:18,460 Any minor things that get fixed, that 553 00:28:18,460 --> 00:28:22,180 need fixing due to sunlight, radiation, chemicals 554 00:28:22,180 --> 00:28:24,040 will be fixed through these methods 555 00:28:24,040 --> 00:28:27,940 to keep that rate, that error rate, in your genome down to, 556 00:28:27,940 --> 00:28:31,330 like, one in a billion or something like that. 557 00:28:31,330 --> 00:28:34,150 All right, I'm just going to flash this up. 558 00:28:34,150 --> 00:28:36,870 I'm going to, very quickly. 559 00:28:36,870 --> 00:28:39,600 I'm going to give you guys a copy of this. 560 00:28:39,600 --> 00:28:42,390 But these are the components that you 561 00:28:42,390 --> 00:28:48,890 want to be able to understand the function of when 562 00:28:48,890 --> 00:28:52,257 thinking about DNA replication. 563 00:28:52,257 --> 00:28:53,840 So you don't have that on your slides, 564 00:28:53,840 --> 00:28:56,215 but we're going to give you a copy so that you can really 565 00:28:56,215 --> 00:28:58,970 make sure that you understand all the moving parts 566 00:28:58,970 --> 00:29:02,250 and how they come together for replication. 567 00:29:02,250 --> 00:29:06,050 Now I want to talk about one last conundrum with DNA, 568 00:29:06,050 --> 00:29:09,350 and that is the issue of telomerase. 569 00:29:15,790 --> 00:29:22,350 And this is particularly critical in eukaryotes 570 00:29:22,350 --> 00:29:26,630 that have linear chromosomes. 571 00:29:31,280 --> 00:29:35,560 Now, if you think about DNA being replicated, 572 00:29:35,560 --> 00:29:42,160 when you look at the two strands of DNA, 573 00:29:42,160 --> 00:29:44,800 as you approach the very end of the chromosome, 574 00:29:44,800 --> 00:29:53,620 you'll do just fine making the copy that's built five primes-- 575 00:29:53,620 --> 00:29:54,500 sorry about this. 576 00:30:09,150 --> 00:30:11,730 You'll do just fine making the copy that 577 00:30:11,730 --> 00:30:14,970 is built in this direction, because it's 578 00:30:14,970 --> 00:30:16,410 the leading strand. 579 00:30:16,410 --> 00:30:17,505 And it's built five prime. 580 00:30:21,310 --> 00:30:23,370 Am I going wrong here? 581 00:30:23,370 --> 00:30:24,930 It's built five prime to three prime. 582 00:30:28,512 --> 00:30:29,720 Can someone help me out here? 583 00:30:29,720 --> 00:30:30,630 I'm losing my mind. 584 00:30:33,630 --> 00:30:38,310 So this piece is built five prime to three prime, 585 00:30:38,310 --> 00:30:41,250 so therefore, this was five prime and three prime. 586 00:30:41,250 --> 00:30:43,560 But then on the other strand, you 587 00:30:43,560 --> 00:30:48,150 have a problem, because you need to put in a primer here 588 00:30:48,150 --> 00:30:50,430 in order to build the other strand. 589 00:30:50,430 --> 00:30:51,880 Does that make sense? 590 00:30:51,880 --> 00:30:54,720 So we've got to have put in that short primer, 591 00:30:54,720 --> 00:30:58,740 because DNA polymerase is will not work otherwise. 592 00:30:58,740 --> 00:31:03,990 And then we need to build this strand of DNA. 593 00:31:03,990 --> 00:31:07,920 All right, so what's the problem here with respect 594 00:31:07,920 --> 00:31:10,650 to these ends? 595 00:31:10,650 --> 00:31:13,266 What's going to happen next? 596 00:31:13,266 --> 00:31:17,900 If it's an RNA primer, what happens next? 597 00:31:17,900 --> 00:31:21,100 We nibble it up, right, with the full intention 598 00:31:21,100 --> 00:31:23,440 of replacing that bit of DNA. 599 00:31:23,440 --> 00:31:26,350 But then what can DNA polymerase do? 600 00:31:26,350 --> 00:31:28,330 It can't do anything, right? 601 00:31:28,330 --> 00:31:32,350 DNA polymerase needs double-stranded DNA 602 00:31:32,350 --> 00:31:35,780 to hold on to so it could fill this gap. 603 00:31:35,780 --> 00:31:42,160 So what happens is every time you replicate DNA, 604 00:31:42,160 --> 00:31:46,250 you end up with a gap, with a small amount 605 00:31:46,250 --> 00:31:47,420 you don't quite copy. 606 00:31:47,420 --> 00:31:49,310 Is everyone following me? 607 00:31:49,310 --> 00:31:50,540 And that's a problem, right? 608 00:31:50,540 --> 00:31:51,957 Because doesn't it mean every time 609 00:31:51,957 --> 00:31:54,950 my cells divide, my genes get a little bit shorter 610 00:31:54,950 --> 00:31:57,830 and a little bit shorter and a little bit shorter? 611 00:31:57,830 --> 00:32:01,880 So there are things in place that help. 612 00:32:01,880 --> 00:32:04,910 One important feature is that, usually, you 613 00:32:04,910 --> 00:32:08,500 don't have important genetic material 614 00:32:08,500 --> 00:32:12,360 at the ends of your genes-- 615 00:32:12,360 --> 00:32:14,840 at the ends of chromosomes, rather. 616 00:32:18,370 --> 00:32:25,190 There's sort of extra DNA that doesn't need to be copied. 617 00:32:25,190 --> 00:32:28,360 But the basic theory, the whole theory about telomerase, 618 00:32:28,360 --> 00:32:31,150 is that for certain types of cells-- 619 00:32:31,150 --> 00:32:36,450 these ascend stem cells and germ cells-- 620 00:32:39,870 --> 00:32:44,070 there is an enzyme that can fill this gap. 621 00:32:44,070 --> 00:32:45,660 It's called telomerase. 622 00:32:52,630 --> 00:32:55,420 So in those cells-- what's special about these types 623 00:32:55,420 --> 00:32:58,340 of cells? 624 00:32:58,340 --> 00:33:00,290 We need to keep them good. 625 00:33:00,290 --> 00:33:03,200 They're what defines your starting DNA. 626 00:33:03,200 --> 00:33:05,810 The stem cells and the germ cells, 627 00:33:05,810 --> 00:33:12,992 like in the sperm and egg, have to have a good copy of DNA. 628 00:33:12,992 --> 00:33:14,700 They can't be getting shorter and shorter 629 00:33:14,700 --> 00:33:17,520 every time a new generation is born. 630 00:33:17,520 --> 00:33:20,470 But once your cells are the somatic cells, 631 00:33:20,470 --> 00:33:22,710 the DNA gets shorter and shorter, 632 00:33:22,710 --> 00:33:26,670 because those cells don't have telomerase. 633 00:33:26,670 --> 00:33:29,890 And this is associated with theories of aging. 634 00:33:29,890 --> 00:33:32,730 So the cells you get, the ones that are finally 635 00:33:32,730 --> 00:33:34,740 the ones in your body, every time 636 00:33:34,740 --> 00:33:38,430 they divide, the ends of the chromosomes 637 00:33:38,430 --> 00:33:39,600 will get a little shorter. 638 00:33:39,600 --> 00:33:42,420 But there's no mechanism to replace those. 639 00:33:42,420 --> 00:33:45,030 And so it's associated with the belief 640 00:33:45,030 --> 00:33:49,170 that, at a certain stage, you've divided the cells enough times. 641 00:33:49,170 --> 00:33:51,210 And then you can't-- 642 00:33:51,210 --> 00:33:55,110 you're actually starting to nibble into important coding 643 00:33:55,110 --> 00:33:56,430 DNA. 644 00:33:56,430 --> 00:33:58,380 Does everybody understand what would 645 00:33:58,380 --> 00:34:00,715 be the significance of that? 646 00:34:00,715 --> 00:34:01,590 Does that make sense? 647 00:34:01,590 --> 00:34:02,227 Yeah? 648 00:34:02,227 --> 00:34:03,688 AUDIENCE: So once the [INAUDIBLE] 649 00:34:03,688 --> 00:34:05,743 are gone, is this [INAUDIBLE] able to divide? 650 00:34:05,743 --> 00:34:06,660 BARBARA IMPERIALI: No. 651 00:34:06,660 --> 00:34:11,969 So the telomerase keeps the DNA in those types 652 00:34:11,969 --> 00:34:14,850 of cells in good condition. 653 00:34:14,850 --> 00:34:18,750 In the cells that divide daily, and your body wants-- 654 00:34:18,750 --> 00:34:20,100 the somatic cells. 655 00:34:20,100 --> 00:34:22,110 You will keep on shortening, but they'll just 656 00:34:22,110 --> 00:34:24,245 be more mistakes, basically. 657 00:34:24,245 --> 00:34:25,620 And those are the sorts of things 658 00:34:25,620 --> 00:34:28,320 that would be associated with an organism that's 659 00:34:28,320 --> 00:34:31,020 growing to a certain age, because it's just 660 00:34:31,020 --> 00:34:32,179 a certain number of cells. 661 00:34:32,179 --> 00:34:34,380 So the cells may not divide, or there 662 00:34:34,380 --> 00:34:39,070 may be mistakes in certain parts of the coding genome. 663 00:34:39,070 --> 00:34:40,489 Any other questions? 664 00:34:40,489 --> 00:34:46,320 OK, so telomerase was also another important discovery 665 00:34:46,320 --> 00:34:48,810 that was awarded a Nobel Prize. 666 00:34:48,810 --> 00:34:50,520 And this gives you details. 667 00:34:50,520 --> 00:34:54,420 So the telomerase protects the genetic information 668 00:34:54,420 --> 00:34:58,470 on every cell division, though you 669 00:34:58,470 --> 00:35:00,940 will lose a little bit of genetic information. 670 00:35:00,940 --> 00:35:02,820 So it limits the number of divisions 671 00:35:02,820 --> 00:35:06,180 a cell can make in a lifetime. 672 00:35:06,180 --> 00:35:08,790 All right, so we're going to move on now. 673 00:35:08,790 --> 00:35:10,750 And I've spent quite a bit of time on this, 674 00:35:10,750 --> 00:35:13,410 but I want to guarantee you that now, 675 00:35:13,410 --> 00:35:16,320 as we move forward to transcription, 676 00:35:16,320 --> 00:35:18,810 there's a few simplifications that we can make 677 00:35:18,810 --> 00:35:21,670 in the story of transcription. 678 00:35:25,650 --> 00:35:28,230 So moving on. 679 00:35:28,230 --> 00:35:29,340 All right. 680 00:35:29,340 --> 00:35:31,050 So what have we done so far? 681 00:35:31,050 --> 00:35:33,100 We've seen replication. 682 00:35:33,100 --> 00:35:39,890 Now we're moving to the process of transcription. 683 00:35:47,490 --> 00:35:50,090 So when you transcribe something, 684 00:35:50,090 --> 00:35:52,950 you're basically making a copy of something, 685 00:35:52,950 --> 00:35:55,520 but in a slightly different format. 686 00:35:55,520 --> 00:35:58,970 So, for example, if you're transcribing from handwritten 687 00:35:58,970 --> 00:36:01,940 to a typed version, you go from something 688 00:36:01,940 --> 00:36:04,460 that's in the script to something that's typed. 689 00:36:04,460 --> 00:36:08,130 It has the same content, but it's in a different format. 690 00:36:08,130 --> 00:36:10,460 So this is what the process is called 691 00:36:10,460 --> 00:36:21,030 when you convert DNA into RNA. 692 00:36:21,030 --> 00:36:24,090 And very specifically, this is part 693 00:36:24,090 --> 00:36:29,020 of the process to make what's known as the messenger RNA. 694 00:36:29,020 --> 00:36:33,420 The first phase of transcription in eukaryotic cells 695 00:36:33,420 --> 00:36:38,050 gets us to a pre-messenger RNA. 696 00:36:38,050 --> 00:36:39,960 So there's a little bit more needs 697 00:36:39,960 --> 00:36:43,020 to be done to it before it can leave the nucleus 698 00:36:43,020 --> 00:36:45,750 to encode protein translation. 699 00:36:45,750 --> 00:36:47,670 But in bacteria, you're basically 700 00:36:47,670 --> 00:36:51,540 just going straight from the DNA to the messenger RNA. 701 00:36:51,540 --> 00:36:53,940 At the beginning of the next class, 702 00:36:53,940 --> 00:36:59,160 we will also talk about going from the pre-messenger RNA 703 00:36:59,160 --> 00:37:00,620 to the messenger RNA. 704 00:37:00,620 --> 00:37:02,100 And let's take a look at the cell 705 00:37:02,100 --> 00:37:06,900 up here, where what we're focusing in on here 706 00:37:06,900 --> 00:37:14,570 is the process whereby we're copying that DNA. 707 00:37:14,570 --> 00:37:17,420 I have no idea why this is really being 708 00:37:17,420 --> 00:37:19,760 monstrously behaved like that. 709 00:37:19,760 --> 00:37:22,850 I'm done, done with these gizmos. 710 00:37:22,850 --> 00:37:26,450 The process whereby the double-stranded DNA opens up 711 00:37:26,450 --> 00:37:29,270 a little bit and we make that pre-messenger RNA 712 00:37:29,270 --> 00:37:30,470 copy, all right? 713 00:37:30,470 --> 00:37:32,570 So I want you to think back to the processes 714 00:37:32,570 --> 00:37:35,250 that we learned about for translation. 715 00:37:35,250 --> 00:37:37,040 And now we're going to move forward 716 00:37:37,040 --> 00:37:39,470 to take a look at transcription. 717 00:37:39,470 --> 00:37:41,770 And frankly, it's a lot simpler. 718 00:37:41,770 --> 00:37:45,097 So let's just look at the players in transcription. 719 00:37:45,097 --> 00:37:46,930 And you've got a copy of this in your notes, 720 00:37:46,930 --> 00:37:49,650 so I don't need to necessarily put it all on the board. 721 00:37:49,650 --> 00:37:55,280 So in DNA, remember, we had a A, G, C, T. We have a deoxyribose, 722 00:37:55,280 --> 00:38:01,010 and it's mostly used as hereditary genetic information. 723 00:38:01,010 --> 00:38:04,790 But in RNA, we're making a new copy 724 00:38:04,790 --> 00:38:08,360 of the DNA, where we use slightly different building 725 00:38:08,360 --> 00:38:09,290 blocks-- 726 00:38:09,290 --> 00:38:12,200 A, G, C, U. U instead of T. Plus, 727 00:38:12,200 --> 00:38:14,510 there are some modified bases that 728 00:38:14,510 --> 00:38:18,770 occur in some of the types of RNA, and the sugar is a ribose. 729 00:38:18,770 --> 00:38:22,190 So the first main thing about the RNA copy 730 00:38:22,190 --> 00:38:26,900 relative to the DNA copy is that ribose/deoxyribose difference. 731 00:38:26,900 --> 00:38:29,090 What's quite remarkable is that when 732 00:38:29,090 --> 00:38:34,620 you have two deoxyribose in your DNA, it's nice and stable. 733 00:38:34,620 --> 00:38:36,740 We need it to be nice and stable. 734 00:38:36,740 --> 00:38:37,700 It's our genome. 735 00:38:37,700 --> 00:38:40,400 We can't let our genome be falling apart 736 00:38:40,400 --> 00:38:42,800 as we're sort of walking down the street. 737 00:38:42,800 --> 00:38:47,960 In contrast, when RNA is used, it's much more transient. 738 00:38:47,960 --> 00:38:51,680 We make a messenger RNA copy of part of DNA 739 00:38:51,680 --> 00:38:53,930 to move forward to make proteins, 740 00:38:53,930 --> 00:38:57,240 but we don't need that to stick around forever. 741 00:38:57,240 --> 00:39:00,620 And when you have the ribose with the two hydroxyls, a two 742 00:39:00,620 --> 00:39:03,590 and three, it's a much more fragile material. 743 00:39:03,590 --> 00:39:09,020 It is a transient message, and it gets degraded quite quickly. 744 00:39:09,020 --> 00:39:11,060 So that difference in the sugar really 745 00:39:11,060 --> 00:39:13,640 dictates the stability there. 746 00:39:13,640 --> 00:39:17,960 RNA is found in a lot of polymers, biopolymers. 747 00:39:17,960 --> 00:39:22,130 We'll talk we'll focus mostly on the messenger RNA today. 748 00:39:22,130 --> 00:39:25,400 It's less than 1% of the DNA. 749 00:39:25,400 --> 00:39:28,250 And then on Friday, we'll be talking about the transfer 750 00:39:28,250 --> 00:39:30,680 RNA and the ribosomal RNA. 751 00:39:30,680 --> 00:39:32,570 So we're really going to focus in 752 00:39:32,570 --> 00:39:36,920 right now on the messenger RNA. 753 00:39:36,920 --> 00:39:41,350 And the one thing about RNA structures I'll elaborate later 754 00:39:41,350 --> 00:39:43,550 is they have very different structures 755 00:39:43,550 --> 00:39:46,700 to canonical DNA, which adopts the double-stranded, 756 00:39:46,700 --> 00:39:48,770 anti-parallel structure. 757 00:39:48,770 --> 00:39:54,380 RNA structures are much more like folded protein structures, 758 00:39:54,380 --> 00:39:56,840 where there may be sections of base pairing, 759 00:39:56,840 --> 00:39:58,790 but there'll also be lots of loops 760 00:39:58,790 --> 00:40:00,840 and different characteristics. 761 00:40:00,840 --> 00:40:05,330 So even the ribose structure makes a difference 762 00:40:05,330 --> 00:40:08,600 in the stability of the double stranded structure 763 00:40:08,600 --> 00:40:12,140 and encourages a lot more of these unusual structures, which 764 00:40:12,140 --> 00:40:14,990 is really why people have a lot of faith 765 00:40:14,990 --> 00:40:20,500 in the theories about the RNA world. 766 00:40:20,500 --> 00:40:24,720 OK, so let's look at DNA polymerase, RNA polymerase. 767 00:40:24,720 --> 00:40:26,960 So here's all the good news that we'll 768 00:40:26,960 --> 00:40:29,000 be able to describe to you. 769 00:40:29,000 --> 00:40:32,630 So when you copy DNA, you copy all of it. 770 00:40:32,630 --> 00:40:35,180 When you copy RNA, you only copy-- 771 00:40:35,180 --> 00:40:37,940 when you make the copy of messenger RNA, 772 00:40:37,940 --> 00:40:41,600 you only copy about 1.5% of the genome. 773 00:40:41,600 --> 00:40:44,060 So you do not copy the entire thing. 774 00:40:44,060 --> 00:40:48,710 So the process is much more restricted to sections of DNA 775 00:40:48,710 --> 00:40:49,940 that need to be copied. 776 00:40:49,940 --> 00:40:53,780 And we'll talk about the features of the DNA that 777 00:40:53,780 --> 00:40:55,110 tell you about that later. 778 00:40:58,640 --> 00:41:01,820 Here's the important details. 779 00:41:01,820 --> 00:41:06,410 So in eukaryotes, transcription happens in the nucleus. 780 00:41:06,410 --> 00:41:18,170 And the key enzyme involved is RNA polymerase, RNA pol. 781 00:41:18,170 --> 00:41:22,750 And it has very different features to DNA polymerase, 782 00:41:22,750 --> 00:41:25,380 but there are two big things that are different. 783 00:41:25,380 --> 00:41:32,830 It includes its own helicase. 784 00:41:32,830 --> 00:41:34,690 So you remember, with replication, we 785 00:41:34,690 --> 00:41:37,690 needed a DNA polymerase and a helicase. 786 00:41:37,690 --> 00:41:40,030 RNA polymerase is much smarter than that. 787 00:41:40,030 --> 00:41:44,200 It actually includes both functions within its structure. 788 00:41:44,200 --> 00:41:49,660 So it's an RNA polymerase that grows the new nucleotide 789 00:41:49,660 --> 00:41:50,860 five prime to three prime. 790 00:41:50,860 --> 00:41:53,620 But it also has a built-in helicase, 791 00:41:53,620 --> 00:41:56,440 so that's an advantage. 792 00:41:56,440 --> 00:42:06,600 It still grows the messenger RNA five prime to three prime, 793 00:42:06,600 --> 00:42:10,050 but it uses the different nucleotide triphosphate 794 00:42:10,050 --> 00:42:10,950 building blocks. 795 00:42:10,950 --> 00:42:12,690 Or one of them is different. 796 00:42:12,690 --> 00:42:16,965 So UTP, ATP. 797 00:42:21,890 --> 00:42:26,330 So remember, the U replaces the T in RNA, 798 00:42:26,330 --> 00:42:28,250 so that's one key difference. 799 00:42:32,750 --> 00:42:35,430 It includes a helicase activity. 800 00:42:35,430 --> 00:42:38,850 And the other really neat thing, because it's 801 00:42:38,850 --> 00:42:42,360 such a complication in replication, 802 00:42:42,360 --> 00:42:47,680 is it doesn't require a primer. 803 00:42:53,770 --> 00:42:56,980 That is why even when we were replicating the DNA, 804 00:42:56,980 --> 00:42:59,530 we were using RNA polymerase to make 805 00:42:59,530 --> 00:43:02,080 those little pieces of primers, because it 806 00:43:02,080 --> 00:43:04,900 didn't need a priming sequence. 807 00:43:04,900 --> 00:43:07,660 So there's really fundamental differences 808 00:43:07,660 --> 00:43:09,700 about the RNA polymerase. 809 00:43:09,700 --> 00:43:12,220 And then the other thing is that only one of the two 810 00:43:12,220 --> 00:43:14,650 strands of DNA is transcribed. 811 00:43:14,650 --> 00:43:17,950 And in a moment, or maybe the beginning of next class, 812 00:43:17,950 --> 00:43:21,580 we'll judge how we can understand 813 00:43:21,580 --> 00:43:25,510 which sequence is transcribed. 814 00:43:25,510 --> 00:43:28,660 And then, obviously, the messenger RNA 815 00:43:28,660 --> 00:43:31,750 is a complementary sequence to the sequence of DNA 816 00:43:31,750 --> 00:43:34,560 that is being copied. 817 00:43:34,560 --> 00:43:39,180 Finally, only part of the DNA is transcribed, 818 00:43:39,180 --> 00:43:42,090 under unlike the process of replication. 819 00:43:42,090 --> 00:43:44,340 All right, so you can see already 820 00:43:44,340 --> 00:43:46,740 that there are a lot of simplifications 821 00:43:46,740 --> 00:43:49,680 in transcription that we did not have the advantage 822 00:43:49,680 --> 00:43:51,150 of in replication. 823 00:43:51,150 --> 00:43:54,750 So the helicase activity and the primary issue 824 00:43:54,750 --> 00:43:59,260 are two key features that make life a lot simpler. 825 00:43:59,260 --> 00:44:03,630 I just wanted to show you this small detail about RNA 826 00:44:03,630 --> 00:44:05,430 polymerase. 827 00:44:05,430 --> 00:44:08,130 There are a lot of natural products out there 828 00:44:08,130 --> 00:44:12,330 that are known to be inhibitors of vital processes, 829 00:44:12,330 --> 00:44:18,540 and one that caught my eye is the small molecules that 830 00:44:18,540 --> 00:44:21,960 are found in mushrooms, the really toxic mushrooms, when 831 00:44:21,960 --> 00:44:22,960 you see some of these. 832 00:44:22,960 --> 00:44:26,380 In fact, never eat a mushroom that you don't know 833 00:44:26,380 --> 00:44:28,380 and you know where it came from, because there's 834 00:44:28,380 --> 00:44:29,400 problems with them. 835 00:44:29,400 --> 00:44:31,170 Because a lot of these mushrooms include 836 00:44:31,170 --> 00:44:33,150 potent natural products. 837 00:44:33,150 --> 00:44:35,250 And in fact, there's a compound known 838 00:44:35,250 --> 00:44:37,830 as amanitin, alpha-amanitin, and it's 839 00:44:37,830 --> 00:44:42,360 found in certain mushrooms known as either the Death Cap 840 00:44:42,360 --> 00:44:44,423 or Destroying Angel mushrooms. 841 00:44:44,423 --> 00:44:45,840 So you could tell from their names 842 00:44:45,840 --> 00:44:47,400 that they are a real problem. 843 00:44:47,400 --> 00:44:52,500 And what the amanitin does is it actually interferes directly 844 00:44:52,500 --> 00:44:58,610 with RNA polymerase by acting as an allosteric inhibitor of RNA 845 00:44:58,610 --> 00:45:01,520 polymerase and locking it into a closed state 846 00:45:01,520 --> 00:45:03,860 so it can't keep on transcribing. 847 00:45:03,860 --> 00:45:07,240 So I thought this was very interesting, incredibly. 848 00:45:07,240 --> 00:45:12,770 Tiny, tiny, tiny, tiny doses will arrest transcription 849 00:45:12,770 --> 00:45:17,250 and cause dire consequences. 850 00:45:17,250 --> 00:45:20,780 So I think what's very interesting is that it's 851 00:45:20,780 --> 00:45:22,130 an allosteric inhibitor. 852 00:45:22,130 --> 00:45:23,560 It's very potent. 853 00:45:23,560 --> 00:45:26,210 What it does is it seals the polymerase 854 00:45:26,210 --> 00:45:29,060 in a locked, closed state that it can't move forward 855 00:45:29,060 --> 00:45:31,130 for transcription. 856 00:45:31,130 --> 00:45:34,280 Now, finally, a couple of points. 857 00:45:34,280 --> 00:45:37,220 When we decide that a portion of gene 858 00:45:37,220 --> 00:45:40,340 is going to be transcribed, there 859 00:45:40,340 --> 00:45:43,010 are a lot of mechanisms in place to identify 860 00:45:43,010 --> 00:45:44,810 the portion of that gene. 861 00:45:44,810 --> 00:45:46,550 And one of the key things that is known 862 00:45:46,550 --> 00:45:50,300 is that there are what are called promoter sites, which 863 00:45:50,300 --> 00:45:52,730 are actually upstream of the portion of gene 864 00:45:52,730 --> 00:45:57,860 that's to be transcribed, where you recruit a bunch of proteins 865 00:45:57,860 --> 00:46:02,930 that actually park down on the double-stranded DNA and then, 866 00:46:02,930 --> 00:46:07,350 at the end of the day, recruit the RNA polymerase. 867 00:46:07,350 --> 00:46:09,440 So all that extra genome, some of it 868 00:46:09,440 --> 00:46:12,530 is not transcribed into messenger RNA 869 00:46:12,530 --> 00:46:14,810 for making proteins, but it's part 870 00:46:14,810 --> 00:46:18,500 of an area of the gene that gets recognized 871 00:46:18,500 --> 00:46:21,350 by all of the proteins that collaborate 872 00:46:21,350 --> 00:46:23,960 to bring in the RNA polymerase in order 873 00:46:23,960 --> 00:46:26,820 for your RNA to be transcribed. 874 00:46:26,820 --> 00:46:31,520 So what I'm showing you here is a double-stranded DNA with one 875 00:46:31,520 --> 00:46:33,540 of the very common promoters that's 876 00:46:33,540 --> 00:46:37,370 just upstream of the part of the DNA that gets transcribed. 877 00:46:37,370 --> 00:46:41,300 And it's called the TATA box, because it's T-A-T-A sequence. 878 00:46:44,000 --> 00:46:46,670 It's got a complement that looks like it. 879 00:46:46,670 --> 00:46:49,040 And it's shown in pink here. 880 00:46:49,040 --> 00:46:54,440 And then the proteins that bind to the DNA at the TATA box 881 00:46:54,440 --> 00:46:58,670 actually drape over that segment of double-stranded DNA 882 00:46:58,670 --> 00:47:00,800 and then serve as recruitment entities 883 00:47:00,800 --> 00:47:05,120 to bring in all the machinery that's needed for transcription 884 00:47:05,120 --> 00:47:07,140 of the gene beyond it. 885 00:47:07,140 --> 00:47:12,080 So some of the identity of all that extra double-stranded DNA 886 00:47:12,080 --> 00:47:15,350 is actually guide places to guide 887 00:47:15,350 --> 00:47:17,780 where the machinery for transcription 888 00:47:17,780 --> 00:47:23,670 has to park in order for messenger RNA to be formed. 889 00:47:23,670 --> 00:47:25,520 So immediately, you can see we're only 890 00:47:25,520 --> 00:47:31,460 going to transcribe part of this genetic material beyond here. 891 00:47:31,460 --> 00:47:34,550 But we need a whole bunch of genetic material 892 00:47:34,550 --> 00:47:36,410 that's actually just serving as sort 893 00:47:36,410 --> 00:47:39,880 of the runway for the plane landing in the right position, 894 00:47:39,880 --> 00:47:40,760 all right? 895 00:47:40,760 --> 00:47:47,880 So I'm going to put up a puzzle that you can think about. 896 00:47:47,880 --> 00:47:49,880 And then we'll start with these at the beginning 897 00:47:49,880 --> 00:47:52,460 of the next class, because I don't want to rush them. 898 00:47:52,460 --> 00:47:55,760 When you decide to transcribe a gene-- 899 00:47:55,760 --> 00:47:58,640 let's say you've got a promoter site here-- 900 00:47:58,640 --> 00:48:00,410 the thing that I want you to think about 901 00:48:00,410 --> 00:48:03,220 is, which strand would you transcribe? 902 00:48:03,220 --> 00:48:05,390 And what's the logic behind this? 903 00:48:05,390 --> 00:48:07,340 And then we'll just do a recap on this 904 00:48:07,340 --> 00:48:08,900 at the beginning of the next class, 905 00:48:08,900 --> 00:48:10,650 because I just want you to think about it. 906 00:48:10,650 --> 00:48:14,010 Because a lot of the information you need is directly here. 907 00:48:14,010 --> 00:48:14,510 Hi there. 908 00:48:14,510 --> 00:48:15,850 We're just wrapping up. 909 00:48:15,850 --> 00:48:16,410 OK? 910 00:48:16,410 --> 00:48:18,550 So that's it for today.