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Format: Layer, Start, End, Style, Name, MarginL, MarginR, MarginV, Effect, Text
Dialogue: 0,0:00:00.24,0:00:03.72,Default,,0000,0000,0000,,- My name is Bob Tjian,\NI'm a Professor of MCB
Dialogue: 0,0:00:03.72,0:00:06.00,Default,,0000,0000,0000,,at the University of\NCalifornia at Berkeley,
Dialogue: 0,0:00:06.00,0:00:10.02,Default,,0000,0000,0000,,and I'm also serving as the\NPresident of the Howard Hughes.
Dialogue: 0,0:00:10.02,0:00:13.44,Default,,0000,0000,0000,,I'm going to spend the\Nnext 25 or 30 minutes
Dialogue: 0,0:00:13.44,0:00:15.87,Default,,0000,0000,0000,,telling you about some fundamentals
Dialogue: 0,0:00:15.87,0:00:19.62,Default,,0000,0000,0000,,of one of the most important\Nmolecular processes
Dialogue: 0,0:00:19.62,0:00:23.49,Default,,0000,0000,0000,,in living cells, which is\Nthe expression of genes
Dialogue: 0,0:00:23.49,0:00:25.59,Default,,0000,0000,0000,,through a process called transcription.
Dialogue: 0,0:00:26.61,0:00:31.61,Default,,0000,0000,0000,,Now, first, to understand\Nwhat gene expression means,
Dialogue: 0,0:00:32.19,0:00:36.36,Default,,0000,0000,0000,,you have to have a sense\Nof what we tend to refer to
Dialogue: 0,0:00:36.36,0:00:39.90,Default,,0000,0000,0000,,in the field as a central\Ndogma of molecular biology.
Dialogue: 0,0:00:39.90,0:00:41.28,Default,,0000,0000,0000,,Another way to think about this
Dialogue: 0,0:00:41.28,0:00:45.88,Default,,0000,0000,0000,,is the flow of biological\Ninformation from DNA,
Dialogue: 0,0:00:46.95,0:00:48.48,Default,,0000,0000,0000,,in other words, our chromosomes,
Dialogue: 0,0:00:48.48,0:00:50.52,Default,,0000,0000,0000,,which every cell has its compliment,
Dialogue: 0,0:00:52.02,0:00:56.73,Default,,0000,0000,0000,,to be transcribed into a\Nsister molecule called RNA.
Dialogue: 0,0:00:56.73,0:00:59.94,Default,,0000,0000,0000,,So, this process of\Nconverting DNA into RNA
Dialogue: 0,0:00:59.94,0:01:01.80,Default,,0000,0000,0000,,is called transcription,
Dialogue: 0,0:01:01.80,0:01:05.37,Default,,0000,0000,0000,,and that is the topic of this lecture.
Dialogue: 0,0:01:05.37,0:01:08.76,Default,,0000,0000,0000,,This process is very complicated,
Dialogue: 0,0:01:08.76,0:01:11.64,Default,,0000,0000,0000,,as you'll see by the\Nend of my two lectures,
Dialogue: 0,0:01:11.64,0:01:13.26,Default,,0000,0000,0000,,and it is very important
Dialogue: 0,0:01:13.26,0:01:18.21,Default,,0000,0000,0000,,for many, many fundamental\Nprocesses in biology.
Dialogue: 0,0:01:18.21,0:01:20.91,Default,,0000,0000,0000,,So, what I'm gonna\Nspend today's lecture on
Dialogue: 0,0:01:20.91,0:01:24.84,Default,,0000,0000,0000,,is the discovery of a large family
Dialogue: 0,0:01:24.84,0:01:27.03,Default,,0000,0000,0000,,of transcription proteins.
Dialogue: 0,0:01:27.03,0:01:31.38,Default,,0000,0000,0000,,These are factors we call\Nthem that are key molecules
Dialogue: 0,0:01:31.38,0:01:35.37,Default,,0000,0000,0000,,that regulate the use\Nof genetic information
Dialogue: 0,0:01:35.37,0:01:37.86,Default,,0000,0000,0000,,that has been encoded in the genome.
Dialogue: 0,0:01:38.73,0:01:41.67,Default,,0000,0000,0000,,Now, transcription factors or proteins
Dialogue: 0,0:01:41.67,0:01:46.05,Default,,0000,0000,0000,,are involved in many\Nfundamental aspects of biology,
Dialogue: 0,0:01:46.05,0:01:48.27,Default,,0000,0000,0000,,including embryonic development,
Dialogue: 0,0:01:48.27,0:01:51.66,Default,,0000,0000,0000,,cellular differentiation, and cell fate.
Dialogue: 0,0:01:51.66,0:01:55.14,Default,,0000,0000,0000,,In other words, pretty much\Nwhat your cells are doing,
Dialogue: 0,0:01:55.14,0:01:56.40,Default,,0000,0000,0000,,how a tissue works,
Dialogue: 0,0:01:56.40,0:01:59.97,Default,,0000,0000,0000,,and how an organism\Nsurvives and reproduces
Dialogue: 0,0:01:59.97,0:02:03.48,Default,,0000,0000,0000,,is dependent on the\Nprocess of gene expression.
Dialogue: 0,0:02:03.48,0:02:06.87,Default,,0000,0000,0000,,And the first step in this\Nprocess is transcription.
Dialogue: 0,0:02:09.15,0:02:11.76,Default,,0000,0000,0000,,Now, there are many other reasons
Dialogue: 0,0:02:11.76,0:02:14.94,Default,,0000,0000,0000,,why a large group of people and scientists
Dialogue: 0,0:02:14.94,0:02:16.41,Default,,0000,0000,0000,,are interested in transcription,
Dialogue: 0,0:02:16.41,0:02:19.35,Default,,0000,0000,0000,,and another reason is that understanding
Dialogue: 0,0:02:19.35,0:02:21.75,Default,,0000,0000,0000,,the fundamental molecular mechanisms
Dialogue: 0,0:02:21.75,0:02:24.99,Default,,0000,0000,0000,,that controls transcription in humans
Dialogue: 0,0:02:24.99,0:02:26.56,Default,,0000,0000,0000,,or in any other organism
Dialogue: 0,0:02:27.42,0:02:31.80,Default,,0000,0000,0000,,can inform us and teach\Nus about what happens
Dialogue: 0,0:02:31.80,0:02:34.86,Default,,0000,0000,0000,,when something goes wrong,\Nfor example, in diseases.
Dialogue: 0,0:02:34.86,0:02:38.10,Default,,0000,0000,0000,,And I list here just a few diseases
Dialogue: 0,0:02:38.10,0:02:40.74,Default,,0000,0000,0000,,that we could study as a result
Dialogue: 0,0:02:40.74,0:02:43.38,Default,,0000,0000,0000,,of understanding the\Nstructure and function
Dialogue: 0,0:02:43.38,0:02:45.90,Default,,0000,0000,0000,,of these transcription factor proteins
Dialogue: 0,0:02:45.90,0:02:48.24,Default,,0000,0000,0000,,that I'm going to be telling you about.
Dialogue: 0,0:02:48.24,0:02:50.55,Default,,0000,0000,0000,,And of course, the hope\Nis that in understanding
Dialogue: 0,0:02:50.55,0:02:54.78,Default,,0000,0000,0000,,the molecular underpinnings of\Ncomplex diseases like cancer,
Dialogue: 0,0:02:54.78,0:02:57.90,Default,,0000,0000,0000,,diabetes, Parkinson's, and so forth,
Dialogue: 0,0:02:57.90,0:03:01.12,Default,,0000,0000,0000,,that we will be able to develop and use
Dialogue: 0,0:03:02.13,0:03:05.04,Default,,0000,0000,0000,,better, more specific therapeutic drugs
Dialogue: 0,0:03:05.04,0:03:07.89,Default,,0000,0000,0000,,and also to develop more accurate
Dialogue: 0,0:03:07.89,0:03:10.20,Default,,0000,0000,0000,,and rapid diagnostic tools.
Dialogue: 0,0:03:10.20,0:03:11.82,Default,,0000,0000,0000,,So, those are a couple of the reasons
Dialogue: 0,0:03:11.82,0:03:14.07,Default,,0000,0000,0000,,why many of us have spent,
Dialogue: 0,0:03:14.07,0:03:15.96,Default,,0000,0000,0000,,in my case, over 30 years
Dialogue: 0,0:03:15.96,0:03:19.17,Default,,0000,0000,0000,,studying this process of\Ntranscriptional regulation.
Dialogue: 0,0:03:20.52,0:03:23.01,Default,,0000,0000,0000,,Now, to get the whole thing started,
Dialogue: 0,0:03:23.01,0:03:24.36,Default,,0000,0000,0000,,I have to give you a sense
Dialogue: 0,0:03:24.36,0:03:27.18,Default,,0000,0000,0000,,of what the magnitude of the problem is.
Dialogue: 0,0:03:27.18,0:03:29.91,Default,,0000,0000,0000,,So, imagine that one would\Nreally like to understand
Dialogue: 0,0:03:29.91,0:03:34.77,Default,,0000,0000,0000,,how this process of decoding\Nthe genome happens in humans.
Dialogue: 0,0:03:34.77,0:03:35.97,Default,,0000,0000,0000,,So, as you may know,
Dialogue: 0,0:03:35.97,0:03:39.12,Default,,0000,0000,0000,,the human genome has\Nsome 3 billion base pairs
Dialogue: 0,0:03:39.12,0:03:41.40,Default,,0000,0000,0000,,or bits of genetic information,
Dialogue: 0,0:03:41.40,0:03:45.00,Default,,0000,0000,0000,,and that encodes roughly 22,000 genes.
Dialogue: 0,0:03:45.00,0:03:48.12,Default,,0000,0000,0000,,These are stretches of DNA sequence
Dialogue: 0,0:03:48.12,0:03:51.39,Default,,0000,0000,0000,,that encode ultimately a product
Dialogue: 0,0:03:51.39,0:03:55.65,Default,,0000,0000,0000,,that is a protein which actually\Nmakes the cells function.
Dialogue: 0,0:03:55.65,0:03:57.81,Default,,0000,0000,0000,,So, as I already explained to you,
Dialogue: 0,0:03:57.81,0:04:00.72,Default,,0000,0000,0000,,there's this flow of\Nbiological information
Dialogue: 0,0:04:00.72,0:04:04.14,Default,,0000,0000,0000,,where you have to extract the\Ninformation buried in DNA,
Dialogue: 0,0:04:04.14,0:04:05.46,Default,,0000,0000,0000,,convert it into RNA.
Dialogue: 0,0:04:05.46,0:04:07.65,Default,,0000,0000,0000,,And what I'm not gonna\Ntell you about today
Dialogue: 0,0:04:07.65,0:04:10.14,Default,,0000,0000,0000,,is the process of going\Nfrom RNA to protein,
Dialogue: 0,0:04:10.14,0:04:13.95,Default,,0000,0000,0000,,which is a reaction called\Na translational reaction.
Dialogue: 0,0:04:13.95,0:04:17.04,Default,,0000,0000,0000,,I'm going to instead just\Nfocus on the first step
Dialogue: 0,0:04:17.04,0:04:18.72,Default,,0000,0000,0000,,of converting DNA into RNA,
Dialogue: 0,0:04:18.72,0:04:20.62,Default,,0000,0000,0000,,which is the process of transcription.
Dialogue: 0,0:04:22.98,0:04:26.85,Default,,0000,0000,0000,,Now, one of the most amazing results
Dialogue: 0,0:04:26.85,0:04:29.16,Default,,0000,0000,0000,,that we got over the last decade or so
Dialogue: 0,0:04:29.16,0:04:31.89,Default,,0000,0000,0000,,was when the human genome\Nwas entirely sequenced,
Dialogue: 0,0:04:31.89,0:04:34.53,Default,,0000,0000,0000,,the first few that were sequenced,
Dialogue: 0,0:04:34.53,0:04:38.13,Default,,0000,0000,0000,,we realized that actually\Nthe number of genes in humans
Dialogue: 0,0:04:38.13,0:04:42.42,Default,,0000,0000,0000,,is not vastly different\Nfrom many other organisms,
Dialogue: 0,0:04:42.42,0:04:45.69,Default,,0000,0000,0000,,even simple organisms like little worms
Dialogue: 0,0:04:45.69,0:04:47.40,Default,,0000,0000,0000,,or fruit flies and so forth.
Dialogue: 0,0:04:47.40,0:04:51.21,Default,,0000,0000,0000,,That is roughly 22 to 25,000 genes
Dialogue: 0,0:04:51.21,0:04:53.01,Default,,0000,0000,0000,,is all the number of genes
Dialogue: 0,0:04:53.01,0:04:55.59,Default,,0000,0000,0000,,that all of these\Ndifferent organisms have.
Dialogue: 0,0:04:55.59,0:04:58.14,Default,,0000,0000,0000,,And yet, anybody looking at us
Dialogue: 0,0:04:58.14,0:05:02.37,Default,,0000,0000,0000,,versus a little roundworm\Nin the soil or a fruit fly
Dialogue: 0,0:05:02.37,0:05:04.83,Default,,0000,0000,0000,,can tell that we're a\Nmuch more complex organism
Dialogue: 0,0:05:04.83,0:05:06.78,Default,,0000,0000,0000,,with a much bigger brain,
Dialogue: 0,0:05:06.78,0:05:09.81,Default,,0000,0000,0000,,much more complex behavior, and so forth.
Dialogue: 0,0:05:09.81,0:05:11.06,Default,,0000,0000,0000,,So, how does this happen?
Dialogue: 0,0:05:12.09,0:05:16.20,Default,,0000,0000,0000,,Part of the answer to this\Nvery interesting mystery
Dialogue: 0,0:05:16.20,0:05:20.01,Default,,0000,0000,0000,,or paradox lies in the way\Nthat genes are organized
Dialogue: 0,0:05:20.01,0:05:21.69,Default,,0000,0000,0000,,and how they're regulated.
Dialogue: 0,0:05:21.69,0:05:23.52,Default,,0000,0000,0000,,And one of the most striking results
Dialogue: 0,0:05:23.52,0:05:26.31,Default,,0000,0000,0000,,of the genome sequencing\Nproject was to realize
Dialogue: 0,0:05:26.31,0:05:31.20,Default,,0000,0000,0000,,that a vast, vast majority\Nof the DNA in our chromosomes
Dialogue: 0,0:05:31.20,0:05:35.07,Default,,0000,0000,0000,,is actually not coding for\Nspecific gene products,
Dialogue: 0,0:05:35.07,0:05:39.93,Default,,0000,0000,0000,,and that only roughly 3% of\Nthe DNA is actually encoding
Dialogue: 0,0:05:39.93,0:05:41.85,Default,,0000,0000,0000,,let's call those little arrows
Dialogue: 0,0:05:41.85,0:05:44.67,Default,,0000,0000,0000,,that I show you on this purple DNA
Dialogue: 0,0:05:44.67,0:05:46.35,Default,,0000,0000,0000,,are the gene coding regions.
Dialogue: 0,0:05:46.35,0:05:50.16,Default,,0000,0000,0000,,So, you'll notice that there's\Na lot of non-arrow sequences,
Dialogue: 0,0:05:50.16,0:05:52.56,Default,,0000,0000,0000,,which I'll show you in\Nthis next slide as green.
Dialogue: 0,0:05:52.56,0:05:54.87,Default,,0000,0000,0000,,These are non-coding regions.
Dialogue: 0,0:05:54.87,0:05:58.80,Default,,0000,0000,0000,,So, the vast majority, 97%\Nor greater is non-coding,
Dialogue: 0,0:05:58.80,0:06:02.55,Default,,0000,0000,0000,,so what are these other sequences doing?
Dialogue: 0,0:06:02.55,0:06:05.79,Default,,0000,0000,0000,,And of course, it turns\Nout that these sequences
Dialogue: 0,0:06:05.79,0:06:10.02,Default,,0000,0000,0000,,carry very important\Nlittle fragments of DNA,
Dialogue: 0,0:06:10.02,0:06:12.42,Default,,0000,0000,0000,,which we call regulatory sequences.
Dialogue: 0,0:06:12.42,0:06:15.45,Default,,0000,0000,0000,,And these are the sequences\Nthat actually control
Dialogue: 0,0:06:15.45,0:06:19.02,Default,,0000,0000,0000,,whether a gene gets turned on or not.
Dialogue: 0,0:06:19.02,0:06:21.63,Default,,0000,0000,0000,,And I'll be spending much\Nof the next 20 minutes
Dialogue: 0,0:06:21.63,0:06:24.39,Default,,0000,0000,0000,,telling you about how\Nthis process all works
Dialogue: 0,0:06:24.39,0:06:28.50,Default,,0000,0000,0000,,and what these little\Nbits of DNA sequences
Dialogue: 0,0:06:28.50,0:06:32.28,Default,,0000,0000,0000,,actually function to\Ncontrol gene expression.
Dialogue: 0,0:06:34.35,0:06:37.29,Default,,0000,0000,0000,,Now, the other thing that I\Nhave to bring you up to date on
Dialogue: 0,0:06:37.29,0:06:40.98,Default,,0000,0000,0000,,is this mysterious process\Nwe're calling transcription,
Dialogue: 0,0:06:40.98,0:06:43.71,Default,,0000,0000,0000,,which reads double-stranded DNA
Dialogue: 0,0:06:43.71,0:06:45.39,Default,,0000,0000,0000,,and then makes a related molecule,
Dialogue: 0,0:06:45.39,0:06:47.55,Default,,0000,0000,0000,,which is a single-stranded RNA molecule,
Dialogue: 0,0:06:47.55,0:06:49.74,Default,,0000,0000,0000,,which is a informational molecule.
Dialogue: 0,0:06:49.74,0:06:54.30,Default,,0000,0000,0000,,That reaction is catalyzed\Nby a very complex
Dialogue: 0,0:06:54.30,0:06:59.01,Default,,0000,0000,0000,,multi-subunit enzyme\Ncalled RNA polymerase II.
Dialogue: 0,0:06:59.01,0:07:01.29,Default,,0000,0000,0000,,Now, there's the roman\Nnumeral II at the end of this
Dialogue: 0,0:07:01.29,0:07:05.73,Default,,0000,0000,0000,,because there were actually\Nthree enzymes in most mammals,
Dialogue: 0,0:07:06.69,0:07:09.27,Default,,0000,0000,0000,,at least three enzymes that\Ncarry out different processes
Dialogue: 0,0:07:09.27,0:07:11.61,Default,,0000,0000,0000,,and different types of RNA production.
Dialogue: 0,0:07:11.61,0:07:12.63,Default,,0000,0000,0000,,But I'm only gonna tell you
Dialogue: 0,0:07:12.63,0:07:16.38,Default,,0000,0000,0000,,about the ones that make\Nthe classical messenger RNA,
Dialogue: 0,0:07:16.38,0:07:19.35,Default,,0000,0000,0000,,which then ultimately becomes proteins.
Dialogue: 0,0:07:19.35,0:07:22.17,Default,,0000,0000,0000,,So, now one of the things that we learned
Dialogue: 0,0:07:22.17,0:07:25.86,Default,,0000,0000,0000,,early on in the study of mammalian
Dialogue: 0,0:07:25.86,0:07:30.09,Default,,0000,0000,0000,,or other multicellular organism\Ntranscription processes
Dialogue: 0,0:07:30.09,0:07:32.64,Default,,0000,0000,0000,,is that despite the fact that this enzyme
Dialogue: 0,0:07:32.64,0:07:34.89,Default,,0000,0000,0000,,is quite complex in its structure,
Dialogue: 0,0:07:35.88,0:07:39.48,Default,,0000,0000,0000,,it turns out to be an\Nenzyme that's nevertheless
Dialogue: 0,0:07:39.48,0:07:42.45,Default,,0000,0000,0000,,needs a lot of help to do its job.
Dialogue: 0,0:07:42.45,0:07:45.09,Default,,0000,0000,0000,,So, on its own, this RNA polymerase II
Dialogue: 0,0:07:45.09,0:07:48.96,Default,,0000,0000,0000,,cannot tell the difference\Nbetween the non-coding regions
Dialogue: 0,0:07:48.96,0:07:52.47,Default,,0000,0000,0000,,of the genome and places where\Nit's supposed to be coding
Dialogue: 0,0:07:52.47,0:07:56.94,Default,,0000,0000,0000,,or reading to make the\Nappropriate messenger RNAs.
Dialogue: 0,0:07:56.94,0:08:00.48,Default,,0000,0000,0000,,So, this sort of leads you\Nto think that there must be
Dialogue: 0,0:08:00.48,0:08:04.95,Default,,0000,0000,0000,,a number of other factors\Nthat somehow direct
Dialogue: 0,0:08:04.95,0:08:08.49,Default,,0000,0000,0000,,RNA polymerase to the right\Nplace at the right time
Dialogue: 0,0:08:08.49,0:08:11.19,Default,,0000,0000,0000,,in the genome of every cell in your body
Dialogue: 0,0:08:11.19,0:08:14.01,Default,,0000,0000,0000,,so that the right products get made
Dialogue: 0,0:08:14.01,0:08:17.40,Default,,0000,0000,0000,,so each cell in your body\Nis functioning properly.
Dialogue: 0,0:08:18.48,0:08:21.54,Default,,0000,0000,0000,,And this is where things\Nget really interesting
Dialogue: 0,0:08:21.54,0:08:25.17,Default,,0000,0000,0000,,is some 25, 30 years ago,
Dialogue: 0,0:08:25.17,0:08:29.31,Default,,0000,0000,0000,,a number of laboratories took on the job
Dialogue: 0,0:08:29.31,0:08:32.64,Default,,0000,0000,0000,,of hunting for these elusive\Nand, as it turned out,
Dialogue: 0,0:08:32.64,0:08:35.34,Default,,0000,0000,0000,,a specialized protein factors
Dialogue: 0,0:08:35.34,0:08:39.39,Default,,0000,0000,0000,,that recognize these little\Nstretches of DNA sequences
Dialogue: 0,0:08:39.39,0:08:40.41,Default,,0000,0000,0000,,that I've been telling you about
Dialogue: 0,0:08:40.41,0:08:42.21,Default,,0000,0000,0000,,that make up the vast majority
Dialogue: 0,0:08:42.21,0:08:45.42,Default,,0000,0000,0000,,of the non-coding part of the genome.
Dialogue: 0,0:08:45.42,0:08:48.48,Default,,0000,0000,0000,,And how these proteins then can recognize
Dialogue: 0,0:08:48.48,0:08:51.15,Default,,0000,0000,0000,,and ultimately physically interact
Dialogue: 0,0:08:51.15,0:08:53.91,Default,,0000,0000,0000,,with these little bits\Nof genetic information
Dialogue: 0,0:08:53.91,0:08:57.66,Default,,0000,0000,0000,,to then turn genes on or off.
Dialogue: 0,0:08:57.66,0:09:00.06,Default,,0000,0000,0000,,Now, in this lecture,
Dialogue: 0,0:09:00.06,0:09:04.32,Default,,0000,0000,0000,,I can't go into all the details\Nof the types of experiments
Dialogue: 0,0:09:04.32,0:09:07.68,Default,,0000,0000,0000,,or the ranges of experiments\Nthat many, many laboratories
Dialogue: 0,0:09:07.68,0:09:09.33,Default,,0000,0000,0000,,have done over the last two decades
Dialogue: 0,0:09:09.33,0:09:12.51,Default,,0000,0000,0000,,to finally work out this molecular puzzle
Dialogue: 0,0:09:12.51,0:09:14.73,Default,,0000,0000,0000,,of how transcription works.
Dialogue: 0,0:09:14.73,0:09:17.10,Default,,0000,0000,0000,,But I can tell you that\Nthere are fundamentally
Dialogue: 0,0:09:17.10,0:09:19.32,Default,,0000,0000,0000,,two major approaches that have been taken
Dialogue: 0,0:09:19.32,0:09:24.32,Default,,0000,0000,0000,,over the last few decades\Nto kind of get a parts list
Dialogue: 0,0:09:24.51,0:09:27.09,Default,,0000,0000,0000,,of the machinery that decodes the genome
Dialogue: 0,0:09:27.09,0:09:29.64,Default,,0000,0000,0000,,and carries out the\Nprocess of transcription.
Dialogue: 0,0:09:29.64,0:09:32.43,Default,,0000,0000,0000,,One is kind of the old style,
Dialogue: 0,0:09:32.43,0:09:34.95,Default,,0000,0000,0000,,I'll call it bucket biochemistry
Dialogue: 0,0:09:34.95,0:09:38.82,Default,,0000,0000,0000,,or take a live cell, crush it up,
Dialogue: 0,0:09:38.82,0:09:41.64,Default,,0000,0000,0000,,spread out all of its parts\Nand then try to figure out
Dialogue: 0,0:09:41.64,0:09:43.05,Default,,0000,0000,0000,,how to put it back together again,
Dialogue: 0,0:09:43.05,0:09:45.54,Default,,0000,0000,0000,,that's what I call in vitro biochemistry.
Dialogue: 0,0:09:45.54,0:09:47.91,Default,,0000,0000,0000,,And the other one is in vivo genetics
Dialogue: 0,0:09:47.91,0:09:50.88,Default,,0000,0000,0000,,where you effectively use genetic tools,
Dialogue: 0,0:09:50.88,0:09:54.57,Default,,0000,0000,0000,,mutagenesis to go in there\Nand selectively remove
Dialogue: 0,0:09:54.57,0:09:59.10,Default,,0000,0000,0000,,or knock down or knock out\Ncertain genes and gene products
Dialogue: 0,0:09:59.10,0:10:01.62,Default,,0000,0000,0000,,and then ask what is the\Nconsequence on that cell
Dialogue: 0,0:10:01.62,0:10:02.97,Default,,0000,0000,0000,,or that organism?
Dialogue: 0,0:10:02.97,0:10:07.97,Default,,0000,0000,0000,,Both of these technologies\Nare very powerful
Dialogue: 0,0:10:08.25,0:10:12.51,Default,,0000,0000,0000,,and highly complementary,\Nand they continue to be used.
Dialogue: 0,0:10:13.41,0:10:16.41,Default,,0000,0000,0000,,Today, I will focus primarily
Dialogue: 0,0:10:16.41,0:10:18.51,Default,,0000,0000,0000,,on the in vitro biochemical techniques
Dialogue: 0,0:10:18.51,0:10:22.74,Default,,0000,0000,0000,,which led us to the discovery\Nof the first few classes
Dialogue: 0,0:10:22.74,0:10:24.45,Default,,0000,0000,0000,,of transcription factors.
Dialogue: 0,0:10:24.45,0:10:25.80,Default,,0000,0000,0000,,And in subsequent lectures,
Dialogue: 0,0:10:25.80,0:10:29.58,Default,,0000,0000,0000,,we'll go to more recent technologies
Dialogue: 0,0:10:29.58,0:10:32.76,Default,,0000,0000,0000,,that allows us to sort of\Nspeed up this whole process
Dialogue: 0,0:10:32.76,0:10:35.37,Default,,0000,0000,0000,,of identifying key regulatory molecules
Dialogue: 0,0:10:35.37,0:10:37.32,Default,,0000,0000,0000,,and how they work.
Dialogue: 0,0:10:38.79,0:10:42.69,Default,,0000,0000,0000,,So, let's go back to the\Nsort of the basic unit
Dialogue: 0,0:10:42.69,0:10:45.06,Default,,0000,0000,0000,,of gene expression, which is a gene,
Dialogue: 0,0:10:45.06,0:10:48.72,Default,,0000,0000,0000,,here shown in the orange arrow,
Dialogue: 0,0:10:48.72,0:10:51.78,Default,,0000,0000,0000,,and the non-coding\Nsequences surrounding it.
Dialogue: 0,0:10:51.78,0:10:54.51,Default,,0000,0000,0000,,And you'll see that now I've\Nadded a few more elements
Dialogue: 0,0:10:54.51,0:10:56.10,Default,,0000,0000,0000,,to this purple DNA.
Dialogue: 0,0:10:56.10,0:10:59.16,Default,,0000,0000,0000,,You see some symbols, a blue square,
Dialogue: 0,0:10:59.16,0:11:02.58,Default,,0000,0000,0000,,a round circle that's pink,\Nand then a yellow triangle.
Dialogue: 0,0:11:02.58,0:11:06.90,Default,,0000,0000,0000,,Those are just a way for\Nme to graphically represent
Dialogue: 0,0:11:06.90,0:11:08.97,Default,,0000,0000,0000,,the little bits of DNA sequences
Dialogue: 0,0:11:08.97,0:11:11.25,Default,,0000,0000,0000,,that I told you about that\Nare the regulatory sequences.
Dialogue: 0,0:11:11.25,0:11:14.73,Default,,0000,0000,0000,,So, the little round one\Nhappens to very GC-rich,
Dialogue: 0,0:11:14.73,0:11:17.97,Default,,0000,0000,0000,,the triangle one is a classical element
Dialogue: 0,0:11:17.97,0:11:19.20,Default,,0000,0000,0000,,that's called a TATA box,
Dialogue: 0,0:11:19.20,0:11:20.61,Default,,0000,0000,0000,,I'll tell you about a little bit later.
Dialogue: 0,0:11:20.61,0:11:23.55,Default,,0000,0000,0000,,And the blue one is yet\Nanother recognition element.
Dialogue: 0,0:11:23.55,0:11:26.52,Default,,0000,0000,0000,,So, why are we so interested\Nin these little stretches
Dialogue: 0,0:11:26.52,0:11:29.76,Default,,0000,0000,0000,,of nucleic acid sequence in the genome
Dialogue: 0,0:11:29.76,0:11:33.24,Default,,0000,0000,0000,,when it's buried amongst\Nbillions of other sequences?
Dialogue: 0,0:11:33.24,0:11:35.58,Default,,0000,0000,0000,,Well, these individual little sequences
Dialogue: 0,0:11:35.58,0:11:38.73,Default,,0000,0000,0000,,turn out to be very important\Nbecause of where they sit,
Dialogue: 0,0:11:38.73,0:11:42.18,Default,,0000,0000,0000,,you'll notice they're sitting\Nnear the top of the arrow,
Dialogue: 0,0:11:42.18,0:11:45.93,Default,,0000,0000,0000,,and they are recognized\Nby very special proteins
Dialogue: 0,0:11:45.93,0:11:48.30,Default,,0000,0000,0000,,which are the transcription factors.
Dialogue: 0,0:11:48.30,0:11:50.64,Default,,0000,0000,0000,,So, now I've showing you some symbols
Dialogue: 0,0:11:50.64,0:11:53.88,Default,,0000,0000,0000,,with little cutouts which\Nfit into either the square,
Dialogue: 0,0:11:53.88,0:11:56.04,Default,,0000,0000,0000,,the circle, or the triangle.
Dialogue: 0,0:11:56.04,0:11:58.56,Default,,0000,0000,0000,,So, transcription factors,
Dialogue: 0,0:11:58.56,0:12:03.15,Default,,0000,0000,0000,,at least one major family\Nof transcription factors,
Dialogue: 0,0:12:03.15,0:12:06.66,Default,,0000,0000,0000,,are proteins whose\Nthree-dimensional structure
Dialogue: 0,0:12:06.66,0:12:10.11,Default,,0000,0000,0000,,is folded into a shape that\Nallows them to recognize
Dialogue: 0,0:12:10.11,0:12:12.66,Default,,0000,0000,0000,,these short stretches\Nof double-stranded DNA.
Dialogue: 0,0:12:13.53,0:12:15.90,Default,,0000,0000,0000,,In fact, largely through interactions
Dialogue: 0,0:12:15.90,0:12:17.40,Default,,0000,0000,0000,,with the major group of DNA,
Dialogue: 0,0:12:17.40,0:12:20.05,Default,,0000,0000,0000,,and I'll show you a structure\Nof one in a little bit.
Dialogue: 0,0:12:21.33,0:12:24.21,Default,,0000,0000,0000,,So, now it turns out\Nthat there are probably
Dialogue: 0,0:12:24.21,0:12:26.61,Default,,0000,0000,0000,,thousands of these transcription factors
Dialogue: 0,0:12:26.61,0:12:29.01,Default,,0000,0000,0000,,because the number of genes\Nthat we have to control,
Dialogue: 0,0:12:29.01,0:12:33.48,Default,,0000,0000,0000,,as I showed you, is in the\Norder of 20 or 25,000 genes.
Dialogue: 0,0:12:33.48,0:12:37.20,Default,,0000,0000,0000,,And so, it turns out that you\Nneed a pretty large percentage
Dialogue: 0,0:12:37.20,0:12:40.89,Default,,0000,0000,0000,,of the genome devoted to encoding\Nthese regulatory proteins
Dialogue: 0,0:12:40.89,0:12:45.24,Default,,0000,0000,0000,,in order for a complex organism\Nlike ourselves to survive.
Dialogue: 0,0:12:45.24,0:12:47.25,Default,,0000,0000,0000,,Then the other component of this,
Dialogue: 0,0:12:47.25,0:12:49.50,Default,,0000,0000,0000,,let's call it the\Ntranscriptional apparatus,
Dialogue: 0,0:12:49.50,0:12:51.90,Default,,0000,0000,0000,,is, of course, the enzyme\Nthat catalyzes RNA.
Dialogue: 0,0:12:51.90,0:12:56.49,Default,,0000,0000,0000,,And I already told you\Nthat this enzyme on its own
Dialogue: 0,0:12:56.49,0:12:59.37,Default,,0000,0000,0000,,can't tell the difference\Nbetween random DNA sequence
Dialogue: 0,0:12:59.37,0:13:01.44,Default,,0000,0000,0000,,and a gene or a promoter.
Dialogue: 0,0:13:01.44,0:13:05.52,Default,,0000,0000,0000,,These other sequence-specific\NDNA-binding proteins
Dialogue: 0,0:13:05.52,0:13:08.55,Default,,0000,0000,0000,,are the ones that must recruit
Dialogue: 0,0:13:08.55,0:13:11.13,Default,,0000,0000,0000,,or otherwise direct RNA polymerase
Dialogue: 0,0:13:11.13,0:13:15.63,Default,,0000,0000,0000,,to essentially land on the right\Nplace and at the right time
Dialogue: 0,0:13:15.63,0:13:19.29,Default,,0000,0000,0000,,in the genome to turn on\Na certain subset of genes
Dialogue: 0,0:13:19.29,0:13:23.64,Default,,0000,0000,0000,,that are specifically required\Nin a specialized cell type,
Dialogue: 0,0:13:23.64,0:13:26.46,Default,,0000,0000,0000,,whatever cell you happen to be looking at.
Dialogue: 0,0:13:26.46,0:13:30.09,Default,,0000,0000,0000,,So, that is kind of the\Nfirst level of complexity
Dialogue: 0,0:13:30.09,0:13:32.91,Default,,0000,0000,0000,,of sort of informational interactions
Dialogue: 0,0:13:32.91,0:13:35.25,Default,,0000,0000,0000,,between the transcription factors
Dialogue: 0,0:13:35.25,0:13:38.25,Default,,0000,0000,0000,,and the more ubiquitous,
Dialogue: 0,0:13:38.25,0:13:41.85,Default,,0000,0000,0000,,and I would call it promiscuous\NRNA polymerase II enzyme.
Dialogue: 0,0:13:43.56,0:13:44.88,Default,,0000,0000,0000,,Well, as it turns out,
Dialogue: 0,0:13:44.88,0:13:49.02,Default,,0000,0000,0000,,it took several decades to work out
Dialogue: 0,0:13:49.02,0:13:52.77,Default,,0000,0000,0000,,most if not all of the components
Dialogue: 0,0:13:52.77,0:13:56.07,Default,,0000,0000,0000,,of this so-called\Ntranscriptional machinery.
Dialogue: 0,0:13:56.07,0:14:00.81,Default,,0000,0000,0000,,And it turns out in this\Nslide I'm showing you
Dialogue: 0,0:14:00.81,0:14:03.03,Default,,0000,0000,0000,,things are already starting\Nto get more complicated.
Dialogue: 0,0:14:03.03,0:14:04.74,Default,,0000,0000,0000,,So, not only do you have RNA polymerase,
Dialogue: 0,0:14:04.74,0:14:07.80,Default,,0000,0000,0000,,but you have a bunch of other\Nproteins that go by names
Dialogue: 0,0:14:07.80,0:14:10.68,Default,,0000,0000,0000,,like TFIIA, B,
Dialogue: 0,0:14:10.68,0:14:13.35,Default,,0000,0000,0000,,you know, D, E, H, F, and so forth.
Dialogue: 0,0:14:13.35,0:14:16.44,Default,,0000,0000,0000,,So, it looks like there\Nare going to be many,
Dialogue: 0,0:14:16.44,0:14:18.27,Default,,0000,0000,0000,,many proteins that are necessary
Dialogue: 0,0:14:18.27,0:14:21.93,Default,,0000,0000,0000,,to form the transcriptional apparatus.
Dialogue: 0,0:14:21.93,0:14:23.49,Default,,0000,0000,0000,,And then on top of that
Dialogue: 0,0:14:23.49,0:14:26.37,Default,,0000,0000,0000,,you need sequence-specific\NDNA-binding proteins
Dialogue: 0,0:14:26.37,0:14:28.56,Default,,0000,0000,0000,,which are already described to you
Dialogue: 0,0:14:28.56,0:14:32.73,Default,,0000,0000,0000,,to further inform or\Notherwise regulate the process
Dialogue: 0,0:14:32.73,0:14:35.58,Default,,0000,0000,0000,,of when a particular\NRNA polymerase molecule
Dialogue: 0,0:14:35.58,0:14:37.86,Default,,0000,0000,0000,,should be binding to a particular gene.
Dialogue: 0,0:14:37.86,0:14:40.11,Default,,0000,0000,0000,,So, that's the sort of overview,
Dialogue: 0,0:14:40.11,0:14:41.58,Default,,0000,0000,0000,,now let me get into the specifics
Dialogue: 0,0:14:41.58,0:14:45.48,Default,,0000,0000,0000,,and how did we actually discover\Nthese family of proteins.
Dialogue: 0,0:14:45.48,0:14:47.28,Default,,0000,0000,0000,,And it'll be interesting for you to see
Dialogue: 0,0:14:47.28,0:14:51.36,Default,,0000,0000,0000,,how science in this field evolved.
Dialogue: 0,0:14:51.36,0:14:54.21,Default,,0000,0000,0000,,Now, as is often the case
Dialogue: 0,0:14:54.21,0:14:56.85,Default,,0000,0000,0000,,when you first try to tackle\Na very complex problem,
Dialogue: 0,0:14:56.85,0:14:59.46,Default,,0000,0000,0000,,and, of course, we didn't\Nreally know how complex it was
Dialogue: 0,0:14:59.46,0:15:00.78,Default,,0000,0000,0000,,when we began these studies,
Dialogue: 0,0:15:00.78,0:15:03.48,Default,,0000,0000,0000,,but we assumed it might be complicated,
Dialogue: 0,0:15:03.48,0:15:06.66,Default,,0000,0000,0000,,certainly would be more\Ncomplicated than systems
Dialogue: 0,0:15:06.66,0:15:09.15,Default,,0000,0000,0000,,that we had already had some idea about,
Dialogue: 0,0:15:09.15,0:15:13.71,Default,,0000,0000,0000,,for example, in bacteria\Nor in bacteriophages.
Dialogue: 0,0:15:13.71,0:15:17.01,Default,,0000,0000,0000,,We took a lesson from our\Nstudies of bacteriophages
Dialogue: 0,0:15:17.01,0:15:20.43,Default,,0000,0000,0000,,and decided that to begin to dissect
Dialogue: 0,0:15:20.43,0:15:22.08,Default,,0000,0000,0000,,the molecular complexities
Dialogue: 0,0:15:22.08,0:15:24.75,Default,,0000,0000,0000,,of the transcription\Nprocess in animal cells,
Dialogue: 0,0:15:24.75,0:15:26.85,Default,,0000,0000,0000,,we should start with viruses
Dialogue: 0,0:15:26.85,0:15:30.84,Default,,0000,0000,0000,,because we knew that viruses\Nwill enter these host cells,
Dialogue: 0,0:15:30.84,0:15:33.99,Default,,0000,0000,0000,,these complex cells that\Nwe ultimately want to study
Dialogue: 0,0:15:33.99,0:15:36.48,Default,,0000,0000,0000,,and have to use the\Nsame molecular machinery
Dialogue: 0,0:15:36.48,0:15:38.58,Default,,0000,0000,0000,,to transcribe their genes
Dialogue: 0,0:15:38.58,0:15:41.64,Default,,0000,0000,0000,,as the host mammalian cell would do.
Dialogue: 0,0:15:41.64,0:15:43.89,Default,,0000,0000,0000,,So, this was kind of a trick
Dialogue: 0,0:15:43.89,0:15:46.98,Default,,0000,0000,0000,,or a way to look at a molecular window
Dialogue: 0,0:15:46.98,0:15:49.71,Default,,0000,0000,0000,,into a complex system\Nand try to simplify it.
Dialogue: 0,0:15:49.71,0:15:51.06,Default,,0000,0000,0000,,And in our case,
Dialogue: 0,0:15:51.06,0:15:54.51,Default,,0000,0000,0000,,the early studies of the\Nlate '70s and early '80s
Dialogue: 0,0:15:54.51,0:15:55.92,Default,,0000,0000,0000,,involved very simple,
Dialogue: 0,0:15:55.92,0:15:58.59,Default,,0000,0000,0000,,one of these simplest\Ndouble-stranded DNA viruses
Dialogue: 0,0:15:58.59,0:16:00.84,Default,,0000,0000,0000,,called Simian virus 40.
Dialogue: 0,0:16:00.84,0:16:03.33,Default,,0000,0000,0000,,And Simian virus 40, of\Ncourse, is a monkey virus,
Dialogue: 0,0:16:03.33,0:16:06.45,Default,,0000,0000,0000,,which was nice because\Nit's very close to humans
Dialogue: 0,0:16:06.45,0:16:07.89,Default,,0000,0000,0000,,and many things that we could learn
Dialogue: 0,0:16:07.89,0:16:10.77,Default,,0000,0000,0000,,about the way this virus uses its host,
Dialogue: 0,0:16:10.77,0:16:13.14,Default,,0000,0000,0000,,which are monkey cells, to replicate
Dialogue: 0,0:16:13.14,0:16:16.44,Default,,0000,0000,0000,,and to express their RNAs and genes
Dialogue: 0,0:16:16.44,0:16:20.37,Default,,0000,0000,0000,,would be applicable to our\Nstudies of humans, as you'll see.
Dialogue: 0,0:16:20.37,0:16:23.19,Default,,0000,0000,0000,,And this virus was one of the first
Dialogue: 0,0:16:23.19,0:16:27.93,Default,,0000,0000,0000,,whose DNA, its double-stranded\NDNA of about 5,000 base pairs
Dialogue: 0,0:16:27.93,0:16:29.31,Default,,0000,0000,0000,,was fully sequenced.
Dialogue: 0,0:16:29.31,0:16:32.67,Default,,0000,0000,0000,,This was long before a\Nrapid modern day sequencing
Dialogue: 0,0:16:32.67,0:16:35.58,Default,,0000,0000,0000,,was available, so this gave\Nus a very powerful tool.
Dialogue: 0,0:16:35.58,0:16:38.34,Default,,0000,0000,0000,,It basically allowed us to\Nlook at the entire genome
Dialogue: 0,0:16:38.34,0:16:41.22,Default,,0000,0000,0000,,of this virus, which\Nwas tiny by comparison,
Dialogue: 0,0:16:41.22,0:16:44.88,Default,,0000,0000,0000,,only 5,243 base pairs.
Dialogue: 0,0:16:44.88,0:16:47.79,Default,,0000,0000,0000,,But just that information\Nwas already very important
Dialogue: 0,0:16:47.79,0:16:49.65,Default,,0000,0000,0000,,'cause it very quickly allowed us,
Dialogue: 0,0:16:49.65,0:16:52.92,Default,,0000,0000,0000,,for example, to map where the genes are.
Dialogue: 0,0:16:52.92,0:16:56.19,Default,,0000,0000,0000,,And one of the genes encoded a protein
Dialogue: 0,0:16:56.19,0:16:57.54,Default,,0000,0000,0000,,called a tumor antigen,
Dialogue: 0,0:16:57.54,0:17:00.21,Default,,0000,0000,0000,,which turns out to be\Na transcription factor.
Dialogue: 0,0:17:00.21,0:17:03.12,Default,,0000,0000,0000,,This then allowed us to get our hands
Dialogue: 0,0:17:03.12,0:17:06.03,Default,,0000,0000,0000,,basically to do biochemistry and genetics
Dialogue: 0,0:17:06.03,0:17:09.39,Default,,0000,0000,0000,,on the very first eukaryotic\Ntranscription factor,
Dialogue: 0,0:17:09.39,0:17:12.21,Default,,0000,0000,0000,,which in this case\Nhappens to be a represser.
Dialogue: 0,0:17:12.21,0:17:15.21,Default,,0000,0000,0000,,That is a protein that\Nwhen it binds the DNA
Dialogue: 0,0:17:15.21,0:17:19.17,Default,,0000,0000,0000,,just the same way as I showed\Nyou for the the model case,
Dialogue: 0,0:17:20.16,0:17:23.91,Default,,0000,0000,0000,,it binds through specific\Nprotein DNA interactions.
Dialogue: 0,0:17:23.91,0:17:26.61,Default,,0000,0000,0000,,But in this case, actually\Nshuts transcription down
Dialogue: 0,0:17:26.61,0:17:27.90,Default,,0000,0000,0000,,rather than turn it up.
Dialogue: 0,0:17:29.58,0:17:33.93,Default,,0000,0000,0000,,In the process of studying\Nthe way that this little virus
Dialogue: 0,0:17:33.93,0:17:36.48,Default,,0000,0000,0000,,when it infects a mammalian cell
Dialogue: 0,0:17:36.48,0:17:39.18,Default,,0000,0000,0000,,uses proteins like T-antigen
Dialogue: 0,0:17:39.18,0:17:42.54,Default,,0000,0000,0000,,to regulate its gene expression,
Dialogue: 0,0:17:42.54,0:17:45.99,Default,,0000,0000,0000,,it became clear that it had\Nto use the host machinery
Dialogue: 0,0:17:45.99,0:17:48.18,Default,,0000,0000,0000,,to do the process.
Dialogue: 0,0:17:48.18,0:17:52.71,Default,,0000,0000,0000,,And that meant that there\Nmust be monkey proteins
Dialogue: 0,0:17:52.71,0:17:55.05,Default,,0000,0000,0000,,that are also involved in activating
Dialogue: 0,0:17:55.05,0:17:57.54,Default,,0000,0000,0000,,or repressing genes of this virus.
Dialogue: 0,0:17:57.54,0:18:00.51,Default,,0000,0000,0000,,And this then led us to\Nthe most important step,
Dialogue: 0,0:18:00.51,0:18:04.17,Default,,0000,0000,0000,,which is to transfer the\Ntechnology we learned about viruses
Dialogue: 0,0:18:04.17,0:18:06.63,Default,,0000,0000,0000,,and how to work with the\Nvirus transcription factor
Dialogue: 0,0:18:06.63,0:18:09.39,Default,,0000,0000,0000,,like T-antigen to the cellular ones.
Dialogue: 0,0:18:09.39,0:18:11.22,Default,,0000,0000,0000,,And I'm gonna give you just one example
Dialogue: 0,0:18:11.22,0:18:13.92,Default,,0000,0000,0000,,of how the simple jump into the host cell
Dialogue: 0,0:18:13.92,0:18:17.82,Default,,0000,0000,0000,,allowed us to discover the first\Nhuman transcription factor.
Dialogue: 0,0:18:17.82,0:18:21.18,Default,,0000,0000,0000,,So, the question that we then asked
Dialogue: 0,0:18:21.18,0:18:25.62,Default,,0000,0000,0000,,back in the early 1980s\Nwas what host molecule
Dialogue: 0,0:18:25.62,0:18:29.31,Default,,0000,0000,0000,,is regulating the expression\Nof transcription of this virus
Dialogue: 0,0:18:29.31,0:18:31.26,Default,,0000,0000,0000,,when the virus is in the host?
Dialogue: 0,0:18:31.26,0:18:33.96,Default,,0000,0000,0000,,And we knew from the DNA\Nsequence of the virus
Dialogue: 0,0:18:33.96,0:18:38.96,Default,,0000,0000,0000,,that there were these six\Nvery GC-rich snippets of DNA
Dialogue: 0,0:18:39.78,0:18:42.24,Default,,0000,0000,0000,,that were regulatory\N'cause if we deleted them,
Dialogue: 0,0:18:42.24,0:18:45.57,Default,,0000,0000,0000,,the virus no longer would\Nexpress the gene of interest.
Dialogue: 0,0:18:45.57,0:18:48.33,Default,,0000,0000,0000,,So, we knew that something\Nwas probably responsible
Dialogue: 0,0:18:48.33,0:18:51.30,Default,,0000,0000,0000,,for recognizing these GC boxes,
Dialogue: 0,0:18:51.30,0:18:53.97,Default,,0000,0000,0000,,and we knew that it wasn't\Na virally encoded gene
Dialogue: 0,0:18:53.97,0:18:56.61,Default,,0000,0000,0000,,because we had tested\Nall of the viral genes
Dialogue: 0,0:18:56.61,0:18:58.95,Default,,0000,0000,0000,,of which there were\Nonly six to begin with.
Dialogue: 0,0:18:58.95,0:19:00.96,Default,,0000,0000,0000,,So, we knew it had to be a host gene
Dialogue: 0,0:19:00.96,0:19:04.74,Default,,0000,0000,0000,,and that led us to a whole, I would say,
Dialogue: 0,0:19:04.74,0:19:07.74,Default,,0000,0000,0000,,family of experiments\Nthat led to the discovery
Dialogue: 0,0:19:07.74,0:19:10.86,Default,,0000,0000,0000,,of sequence-specific mammalian\Ntranscription factors.
Dialogue: 0,0:19:10.86,0:19:13.86,Default,,0000,0000,0000,,And as I said, we could have\Ntaken multiple approaches
Dialogue: 0,0:19:13.86,0:19:16.83,Default,,0000,0000,0000,,to try to address this complicated issue.
Dialogue: 0,0:19:16.83,0:19:18.51,Default,,0000,0000,0000,,I'll just give you one example
Dialogue: 0,0:19:18.51,0:19:20.94,Default,,0000,0000,0000,,of using in vitro biochemistry
Dialogue: 0,0:19:20.94,0:19:24.78,Default,,0000,0000,0000,,to finally get our hands\Non this key sequence
Dialogue: 0,0:19:24.78,0:19:27.30,Default,,0000,0000,0000,,specific human transcription factor,
Dialogue: 0,0:19:27.30,0:19:30.99,Default,,0000,0000,0000,,which, of course, has a\Nhomologue in the monkey.
Dialogue: 0,0:19:31.95,0:19:35.19,Default,,0000,0000,0000,,And the way we did it was very interesting
Dialogue: 0,0:19:35.19,0:19:36.93,Default,,0000,0000,0000,,and simple in retrospect,
Dialogue: 0,0:19:36.93,0:19:39.03,Default,,0000,0000,0000,,and that is recognizing the fact
Dialogue: 0,0:19:39.03,0:19:41.76,Default,,0000,0000,0000,,that whatever this protein was,
Dialogue: 0,0:19:41.76,0:19:44.76,Default,,0000,0000,0000,,it had to have the property of recognizing
Dialogue: 0,0:19:44.76,0:19:49.65,Default,,0000,0000,0000,,those GC boxes that were sitting\Nnext to the the viral gene.
Dialogue: 0,0:19:49.65,0:19:52.20,Default,,0000,0000,0000,,We assume that it must\Nbe a sequence-specific
Dialogue: 0,0:19:52.20,0:19:54.06,Default,,0000,0000,0000,,DNA binding-protein, so all we had to do
Dialogue: 0,0:19:54.06,0:19:57.51,Default,,0000,0000,0000,,was figure out a way to extract proteins
Dialogue: 0,0:19:57.51,0:20:01.11,Default,,0000,0000,0000,,from human cells or monkey cells
Dialogue: 0,0:20:01.11,0:20:04.77,Default,,0000,0000,0000,,and then try to fish out\Nthose specific proteins
Dialogue: 0,0:20:04.77,0:20:06.66,Default,,0000,0000,0000,,out of the many thousands\Nof different proteins
Dialogue: 0,0:20:06.66,0:20:09.72,Default,,0000,0000,0000,,that were in this gamish\Nof cellular extract
Dialogue: 0,0:20:09.72,0:20:12.30,Default,,0000,0000,0000,,that would be responsible\Nfor discriminating
Dialogue: 0,0:20:12.30,0:20:17.10,Default,,0000,0000,0000,,between random DNA sequences\Nand the specific GC box.
Dialogue: 0,0:20:17.10,0:20:20.58,Default,,0000,0000,0000,,And I'll quickly run through\Nsort of the logic behind this.
Dialogue: 0,0:20:20.58,0:20:25.44,Default,,0000,0000,0000,,So, what I'm showing you\Nhere is a solid surface
Dialogue: 0,0:20:25.44,0:20:28.83,Default,,0000,0000,0000,,with DNA coupled to it\Nthat is highly enriched
Dialogue: 0,0:20:28.83,0:20:31.44,Default,,0000,0000,0000,,for the recognition element, the GC box,
Dialogue: 0,0:20:31.44,0:20:33.45,Default,,0000,0000,0000,,which should be the sequence
Dialogue: 0,0:20:33.45,0:20:35.37,Default,,0000,0000,0000,,recognized by the protein of interest.
Dialogue: 0,0:20:35.37,0:20:37.44,Default,,0000,0000,0000,,Now, we had no idea what this\Nprotein was gonna look like,
Dialogue: 0,0:20:37.44,0:20:39.51,Default,,0000,0000,0000,,how many proteins there\Nwere gonna be, and so forth,
Dialogue: 0,0:20:39.51,0:20:42.03,Default,,0000,0000,0000,,but we knew it had to\Nrecognize the GC box.
Dialogue: 0,0:20:42.03,0:20:45.09,Default,,0000,0000,0000,,So, we're gonna try to\Nfish this out of a pool
Dialogue: 0,0:20:45.09,0:20:47.46,Default,,0000,0000,0000,,of many thousands of other proteins.
Dialogue: 0,0:20:47.46,0:20:49.50,Default,,0000,0000,0000,,Now, the the key trick here
Dialogue: 0,0:20:49.50,0:20:52.38,Default,,0000,0000,0000,,was that because all cell extracts
Dialogue: 0,0:20:52.38,0:20:54.87,Default,,0000,0000,0000,,contain not only one DNA binding protein,
Dialogue: 0,0:20:54.87,0:20:56.82,Default,,0000,0000,0000,,but, as I told you, thousands of different
Dialogue: 0,0:20:56.82,0:20:58.41,Default,,0000,0000,0000,,DNA binding proteins.
Dialogue: 0,0:20:58.41,0:21:00.87,Default,,0000,0000,0000,,But most of them, or in fact in our case,
Dialogue: 0,0:21:00.87,0:21:04.95,Default,,0000,0000,0000,,none of the other of several\Nhundred to a thousand proteins
Dialogue: 0,0:21:04.95,0:21:08.79,Default,,0000,0000,0000,,that could bind DNA actually\Nhappen to recognize the GC box,
Dialogue: 0,0:21:08.79,0:21:11.19,Default,,0000,0000,0000,,they just bind other DNA sequences.
Dialogue: 0,0:21:11.19,0:21:14.07,Default,,0000,0000,0000,,So, to kind of favor our protein
Dialogue: 0,0:21:14.07,0:21:16.08,Default,,0000,0000,0000,,being able to bind to our GC box
Dialogue: 0,0:21:16.08,0:21:18.78,Default,,0000,0000,0000,,and not have to compete\Nwith all the other proteins,
Dialogue: 0,0:21:18.78,0:21:22.92,Default,,0000,0000,0000,,what we did was to add non-specific DNA
Dialogue: 0,0:21:22.92,0:21:26.70,Default,,0000,0000,0000,,and mask stoichiometric excess
Dialogue: 0,0:21:26.70,0:21:29.58,Default,,0000,0000,0000,,so that all the other proteins\Nthat wouldn't recognize
Dialogue: 0,0:21:29.58,0:21:33.27,Default,,0000,0000,0000,,the GC box would still have\Nsome partner to hang onto.
Dialogue: 0,0:21:33.27,0:21:34.92,Default,,0000,0000,0000,,And this trick worked very well.
Dialogue: 0,0:21:34.92,0:21:39.84,Default,,0000,0000,0000,,So, having the specific\NDNA on the solid resin
Dialogue: 0,0:21:39.84,0:21:43.71,Default,,0000,0000,0000,,and the non-specific DNA\Nflowing all over the place,
Dialogue: 0,0:21:43.71,0:21:47.85,Default,,0000,0000,0000,,we could capture selectively\Nthe pink molecules here,
Dialogue: 0,0:21:47.85,0:21:50.16,Default,,0000,0000,0000,,which are the GC box recognition ones,
Dialogue: 0,0:21:50.16,0:21:52.56,Default,,0000,0000,0000,,and the blue-green molecules,
Dialogue: 0,0:21:52.56,0:21:56.04,Default,,0000,0000,0000,,of course, predominantly\Nbind to non-specific DNA.
Dialogue: 0,0:21:56.04,0:21:58.47,Default,,0000,0000,0000,,I show you one little\Nblue one on the column
Dialogue: 0,0:21:58.47,0:22:01.29,Default,,0000,0000,0000,,because nothing works\Nperfectly in real science
Dialogue: 0,0:22:01.29,0:22:03.54,Default,,0000,0000,0000,,and tells you that we have\Nto go through this process
Dialogue: 0,0:22:03.54,0:22:07.65,Default,,0000,0000,0000,,iteratively to actually\Nfinally obtain a preparation
Dialogue: 0,0:22:07.65,0:22:11.52,Default,,0000,0000,0000,,that's purely pink molecules\Nwith no green-blue ones.
Dialogue: 0,0:22:11.52,0:22:14.34,Default,,0000,0000,0000,,Well, that turned out\Nto work very, very well.
Dialogue: 0,0:22:14.34,0:22:18.03,Default,,0000,0000,0000,,And that whole process of\Nbiochemical fractionation
Dialogue: 0,0:22:18.03,0:22:23.03,Default,,0000,0000,0000,,followed by a direct affinity\Nsequence-specific DNA resin
Dialogue: 0,0:22:23.73,0:22:28.11,Default,,0000,0000,0000,,gave us the ability to perform\Na biochemical purification
Dialogue: 0,0:22:28.11,0:22:31.62,Default,,0000,0000,0000,,followed by a molecular cloning\Nof the transcription factor
Dialogue: 0,0:22:31.62,0:22:35.04,Default,,0000,0000,0000,,that encodes the protein SP1.
Dialogue: 0,0:22:35.04,0:22:37.23,Default,,0000,0000,0000,,And then we carried out\Na bunch of experiments,
Dialogue: 0,0:22:37.23,0:22:38.58,Default,,0000,0000,0000,,which I'll tell you next,
Dialogue: 0,0:22:38.58,0:22:40.14,Default,,0000,0000,0000,,to show that this protein
Dialogue: 0,0:22:40.14,0:22:42.48,Default,,0000,0000,0000,,actually does activate transcription.
Dialogue: 0,0:22:43.53,0:22:46.38,Default,,0000,0000,0000,,And of course, we went back and\Nwe proved that this protein,
Dialogue: 0,0:22:46.38,0:22:49.17,Default,,0000,0000,0000,,which turned out to be a\Nrather large polypeptide,
Dialogue: 0,0:22:49.17,0:22:52.02,Default,,0000,0000,0000,,can indeed recognize the GC box.
Dialogue: 0,0:22:52.02,0:22:55.50,Default,,0000,0000,0000,,And it doesn't matter if it's\Na GC box from the SV 0 genome
Dialogue: 0,0:22:55.50,0:22:59.46,Default,,0000,0000,0000,,or any other GC box that we\Ncould find in the human genome,
Dialogue: 0,0:22:59.46,0:23:02.37,Default,,0000,0000,0000,,it would find that sequence and bind to it
Dialogue: 0,0:23:02.37,0:23:05.67,Default,,0000,0000,0000,,and then it would generally\Nactivate transcription.
Dialogue: 0,0:23:05.67,0:23:08.28,Default,,0000,0000,0000,,So, this led to the discovery of the first
Dialogue: 0,0:23:08.28,0:23:10.83,Default,,0000,0000,0000,,of a very large family
Dialogue: 0,0:23:10.83,0:23:13.68,Default,,0000,0000,0000,,of sequence-specific DNA-binding proteins.
Dialogue: 0,0:23:13.68,0:23:15.96,Default,,0000,0000,0000,,Now, I told you that\Nthe way these proteins
Dialogue: 0,0:23:15.96,0:23:19.20,Default,,0000,0000,0000,,tend to recognize short DNA sequences
Dialogue: 0,0:23:19.20,0:23:21.90,Default,,0000,0000,0000,,is to interact with DNA\Nthrough the major groove.
Dialogue: 0,0:23:21.90,0:23:23.22,Default,,0000,0000,0000,,And here's a perfect example.
Dialogue: 0,0:23:23.22,0:23:25.23,Default,,0000,0000,0000,,So, the thick blue model there
Dialogue: 0,0:23:25.23,0:23:28.59,Default,,0000,0000,0000,,shows the actual three structures
Dialogue: 0,0:23:28.59,0:23:29.91,Default,,0000,0000,0000,,that are called zinc fingers.
Dialogue: 0,0:23:29.91,0:23:31.47,Default,,0000,0000,0000,,And the reason they're called zinc fingers
Dialogue: 0,0:23:31.47,0:23:34.77,Default,,0000,0000,0000,,is because there are amino\Nacids that are organized
Dialogue: 0,0:23:34.77,0:23:37.86,Default,,0000,0000,0000,,around a center that\Ncontains a zinc molecule
Dialogue: 0,0:23:37.86,0:23:41.19,Default,,0000,0000,0000,,which holds the three-dimensional\Nshape of the polypeptide
Dialogue: 0,0:23:41.19,0:23:43.56,Default,,0000,0000,0000,,in a position just right
Dialogue: 0,0:23:43.56,0:23:45.57,Default,,0000,0000,0000,,for fitting into the\Nmajor groove of the DNA.
Dialogue: 0,0:23:45.57,0:23:47.70,Default,,0000,0000,0000,,And the DNA here is shown in pink,
Dialogue: 0,0:23:47.70,0:23:50.16,Default,,0000,0000,0000,,and you can see that that blue outline
Dialogue: 0,0:23:50.16,0:23:52.71,Default,,0000,0000,0000,,fits right into the\Nmajor groove of the DNA,
Dialogue: 0,0:23:52.71,0:23:54.69,Default,,0000,0000,0000,,but not to the minor groove.
Dialogue: 0,0:23:54.69,0:23:57.54,Default,,0000,0000,0000,,And one of the most important findings
Dialogue: 0,0:23:57.54,0:23:58.83,Default,,0000,0000,0000,,was not only the discovery
Dialogue: 0,0:23:58.83,0:24:00.75,Default,,0000,0000,0000,,of the first human transcription factor,
Dialogue: 0,0:24:00.75,0:24:04.89,Default,,0000,0000,0000,,but the realization that most\Nif not all sequence-specific
Dialogue: 0,0:24:04.89,0:24:06.57,Default,,0000,0000,0000,,DNA-binding transcription factors
Dialogue: 0,0:24:06.57,0:24:09.09,Default,,0000,0000,0000,,have a similar structural motif.
Dialogue: 0,0:24:09.09,0:24:13.17,Default,,0000,0000,0000,,That is to say some structure\Nis built to recognize
Dialogue: 0,0:24:13.17,0:24:15.75,Default,,0000,0000,0000,,sequences in the major groove of DNA.
Dialogue: 0,0:24:15.75,0:24:19.17,Default,,0000,0000,0000,,And these three-dimensional motifs
Dialogue: 0,0:24:19.17,0:24:23.76,Default,,0000,0000,0000,,are recognizable as amino\Nacid sequences in the genome.
Dialogue: 0,0:24:23.76,0:24:27.81,Default,,0000,0000,0000,,So, we can now much more\Nquickly scan the entire sequence
Dialogue: 0,0:24:27.81,0:24:29.76,Default,,0000,0000,0000,,of a genome and identify genes
Dialogue: 0,0:24:29.76,0:24:31.92,Default,,0000,0000,0000,,that are likely to be DNA-binding proteins
Dialogue: 0,0:24:31.92,0:24:34.86,Default,,0000,0000,0000,,as a result of understanding\Nthe structure-function
Dialogue: 0,0:24:34.86,0:24:38.85,Default,,0000,0000,0000,,relationships of these DNA-binding\Nmotifs like zinc fingers.
Dialogue: 0,0:24:39.96,0:24:42.69,Default,,0000,0000,0000,,So, what I'd like to show you now
Dialogue: 0,0:24:42.69,0:24:46.26,Default,,0000,0000,0000,,is that I've only\Nintroduced you to one class
Dialogue: 0,0:24:46.26,0:24:48.21,Default,,0000,0000,0000,,of transcription factors,
Dialogue: 0,0:24:48.21,0:24:51.21,Default,,0000,0000,0000,,which are the sequence-specific-DNA\Nbinding proteins.
Dialogue: 0,0:24:51.21,0:24:53.91,Default,,0000,0000,0000,,Well, I think I gave you a little taste
Dialogue: 0,0:24:53.91,0:24:55.32,Default,,0000,0000,0000,,of the level of complexity
Dialogue: 0,0:24:55.32,0:24:57.06,Default,,0000,0000,0000,,that's probably going to be needed
Dialogue: 0,0:24:57.06,0:24:59.58,Default,,0000,0000,0000,,to be able to build the machine
Dialogue: 0,0:24:59.58,0:25:02.94,Default,,0000,0000,0000,,that's ultimately going\Nto be able to allow you
Dialogue: 0,0:25:02.94,0:25:07.05,Default,,0000,0000,0000,,to transcribe every gene in\Nevery cell of a human body.
Dialogue: 0,0:25:07.05,0:25:10.29,Default,,0000,0000,0000,,So, that turns out to be a\Nmuch more elaborated machine
Dialogue: 0,0:25:10.29,0:25:12.09,Default,,0000,0000,0000,,than what I just showed you.
Dialogue: 0,0:25:12.09,0:25:14.40,Default,,0000,0000,0000,,So, I wanna show you now
Dialogue: 0,0:25:14.40,0:25:16.83,Default,,0000,0000,0000,,what is sort of our\Nstate-of-the-art thinking
Dialogue: 0,0:25:16.83,0:25:20.58,Default,,0000,0000,0000,,about what is actually\Nneeded to build the machinery
Dialogue: 0,0:25:20.58,0:25:25.38,Default,,0000,0000,0000,,at a gene to allow it to be\Nexpressed and transcribed.
Dialogue: 0,0:25:25.38,0:25:27.93,Default,,0000,0000,0000,,And the term I want to introduce you to
Dialogue: 0,0:25:27.93,0:25:30.87,Default,,0000,0000,0000,,is the pre-initiation complex.
Dialogue: 0,0:25:30.87,0:25:33.36,Default,,0000,0000,0000,,And it's pretty much what it says.
Dialogue: 0,0:25:33.36,0:25:35.85,Default,,0000,0000,0000,,It's the complex of multiple subunits
Dialogue: 0,0:25:35.85,0:25:40.85,Default,,0000,0000,0000,,that has to essentially land\Non the promoter of a gene
Dialogue: 0,0:25:40.86,0:25:44.43,Default,,0000,0000,0000,,which will be designated\Nfor later expression.
Dialogue: 0,0:25:45.39,0:25:49.98,Default,,0000,0000,0000,,And this is a process that\Nis probably quite orderly,
Dialogue: 0,0:25:49.98,0:25:52.41,Default,,0000,0000,0000,,that is there's an order\Nof events that happens,
Dialogue: 0,0:25:52.41,0:25:55.08,Default,,0000,0000,0000,,which we, by the way,\Nare not entirely sure
Dialogue: 0,0:25:55.08,0:25:57.33,Default,,0000,0000,0000,,exactly what the order\Nis or even if the order
Dialogue: 0,0:25:57.33,0:25:59.31,Default,,0000,0000,0000,,is the same from one gene to the next,
Dialogue: 0,0:25:59.31,0:26:02.07,Default,,0000,0000,0000,,but we can kind of see where\Nit starts and where it ends up.
Dialogue: 0,0:26:02.07,0:26:03.69,Default,,0000,0000,0000,,And the pathway in between,
Dialogue: 0,0:26:03.69,0:26:06.54,Default,,0000,0000,0000,,I would say is still a little bit murky.
Dialogue: 0,0:26:06.54,0:26:10.29,Default,,0000,0000,0000,,And the story here again starts\Nwith a little snippet of DNA
Dialogue: 0,0:26:10.29,0:26:11.22,Default,,0000,0000,0000,,called the TATA box,
Dialogue: 0,0:26:11.22,0:26:13.56,Default,,0000,0000,0000,,which I already introduced you to briefly.
Dialogue: 0,0:26:13.56,0:26:18.56,Default,,0000,0000,0000,,It's an AT-rich sequence which\Nsits at the five prime end
Dialogue: 0,0:26:18.66,0:26:20.97,Default,,0000,0000,0000,,or the beginning of many\Ngenes, but not all genes,
Dialogue: 0,0:26:20.97,0:26:25.38,Default,,0000,0000,0000,,maybe 20% of the genes might\Ncontain this AT-rich region.
Dialogue: 0,0:26:26.46,0:26:29.85,Default,,0000,0000,0000,,And that AT sequence is the signal
Dialogue: 0,0:26:29.85,0:26:31.50,Default,,0000,0000,0000,,or a landmark, if you like,
Dialogue: 0,0:26:31.50,0:26:33.93,Default,,0000,0000,0000,,for a particular protein to bind to it.
Dialogue: 0,0:26:33.93,0:26:35.85,Default,,0000,0000,0000,,And that protein is called,
Dialogue: 0,0:26:35.85,0:26:38.19,Default,,0000,0000,0000,,not surprisingly, the TATA-binding protein
Dialogue: 0,0:26:38.19,0:26:40.29,Default,,0000,0000,0000,,'cause it's the TATA sequence.
Dialogue: 0,0:26:40.29,0:26:43.62,Default,,0000,0000,0000,,And so, this represents a second class
Dialogue: 0,0:26:43.62,0:26:45.21,Default,,0000,0000,0000,,of transcription factors.
Dialogue: 0,0:26:45.21,0:26:48.24,Default,,0000,0000,0000,,These are not the type that\NI just introduced you to,
Dialogue: 0,0:26:48.24,0:26:50.64,Default,,0000,0000,0000,,which are gonna be\Ndifferent for every gene,
Dialogue: 0,0:26:50.64,0:26:52.20,Default,,0000,0000,0000,,the TATA sequence is present
Dialogue: 0,0:26:52.20,0:26:54.12,Default,,0000,0000,0000,,in a very large number of genes,
Dialogue: 0,0:26:54.12,0:26:56.70,Default,,0000,0000,0000,,so it can't be gene specific,
Dialogue: 0,0:26:56.70,0:26:58.68,Default,,0000,0000,0000,,but it turns out to be very crucial
Dialogue: 0,0:26:58.68,0:27:02.13,Default,,0000,0000,0000,,for our understanding of\Nhow gene regulation works.
Dialogue: 0,0:27:02.13,0:27:06.30,Default,,0000,0000,0000,,So, so you start with\Na TATA-binding protein
Dialogue: 0,0:27:06.30,0:27:07.95,Default,,0000,0000,0000,,finding a TATA box.
Dialogue: 0,0:27:07.95,0:27:10.44,Default,,0000,0000,0000,,We later found out that\Nthe TATA-binding protein
Dialogue: 0,0:27:10.44,0:27:13.92,Default,,0000,0000,0000,,rarely functions on its own\Nand has a bunch of friends
Dialogue: 0,0:27:13.92,0:27:17.01,Default,,0000,0000,0000,,that we call TAFs or\NTBP associated factors.
Dialogue: 0,0:27:17.01,0:27:19.26,Default,,0000,0000,0000,,And now you're talking about an assembly
Dialogue: 0,0:27:19.26,0:27:23.34,Default,,0000,0000,0000,,of multi-subunit complex of\Nalmost a million daltons.
Dialogue: 0,0:27:23.34,0:27:26.07,Default,,0000,0000,0000,,There are somewhere\Nbetween 12 to 15 subunits
Dialogue: 0,0:27:26.07,0:27:27.78,Default,,0000,0000,0000,,in addition to the TATA-binding protein
Dialogue: 0,0:27:27.78,0:27:30.93,Default,,0000,0000,0000,,that make up this little\Ncomplex of proteins
Dialogue: 0,0:27:30.93,0:27:33.39,Default,,0000,0000,0000,,that kind of travels around together.
Dialogue: 0,0:27:33.39,0:27:35.52,Default,,0000,0000,0000,,And this is found in most cell types,
Dialogue: 0,0:27:35.52,0:27:38.76,Default,,0000,0000,0000,,and later on I'll show you\Nin a subsequent lecture
Dialogue: 0,0:27:38.76,0:27:40.59,Default,,0000,0000,0000,,that not every cell type
Dialogue: 0,0:27:40.59,0:27:43.53,Default,,0000,0000,0000,,might have exactly the same\Ncompliment of these subunits,
Dialogue: 0,0:27:43.53,0:27:47.85,Default,,0000,0000,0000,,but many of them have\Nthis prototypic complex.
Dialogue: 0,0:27:47.85,0:27:51.96,Default,,0000,0000,0000,,Is this enough for building\Nthe pre-initiation complex?
Dialogue: 0,0:27:51.96,0:27:54.12,Default,,0000,0000,0000,,Unfortunately not.
Dialogue: 0,0:27:54.12,0:27:57.63,Default,,0000,0000,0000,,It turns out that there\Nare a host of other,
Dialogue: 0,0:27:57.63,0:28:00.03,Default,,0000,0000,0000,,I'll call them ancillary factors
Dialogue: 0,0:28:00.03,0:28:03.33,Default,,0000,0000,0000,,in addition to the multi-subunit\NRNA polymerase itself
Dialogue: 0,0:28:03.33,0:28:08.33,Default,,0000,0000,0000,,that are necessary for you\Nto build up an ensemble
Dialogue: 0,0:28:08.49,0:28:12.18,Default,,0000,0000,0000,,that is necessary to form an active
Dialogue: 0,0:28:12.18,0:28:16.38,Default,,0000,0000,0000,,ready to activate transcriptional\Npre-initiation complex
Dialogue: 0,0:28:16.38,0:28:17.21,Default,,0000,0000,0000,,or the PIC.
Dialogue: 0,0:28:19.89,0:28:23.52,Default,,0000,0000,0000,,And this is kind of the\Npicture we're getting to,
Dialogue: 0,0:28:23.52,0:28:26.16,Default,,0000,0000,0000,,and even this picture\Nwith many, many colors
Dialogue: 0,0:28:26.16,0:28:28.56,Default,,0000,0000,0000,,and many, many different polypeptides,
Dialogue: 0,0:28:28.56,0:28:30.33,Default,,0000,0000,0000,,you know, that adds up to probably greater
Dialogue: 0,0:28:30.33,0:28:33.66,Default,,0000,0000,0000,,than 85 individual proteins
Dialogue: 0,0:28:33.66,0:28:36.96,Default,,0000,0000,0000,,that all have to kind of fit\Ntogether like a jigsaw puzzle.
Dialogue: 0,0:28:36.96,0:28:39.36,Default,,0000,0000,0000,,It's probably not even the whole story,
Dialogue: 0,0:28:39.36,0:28:42.39,Default,,0000,0000,0000,,you'll notice I still have one\Nbig red question mark there
Dialogue: 0,0:28:42.39,0:28:46.98,Default,,0000,0000,0000,,because I think as we begin\Nto study specific cell types
Dialogue: 0,0:28:46.98,0:28:50.28,Default,,0000,0000,0000,,and specific processes\Nlike embryonic development
Dialogue: 0,0:28:50.28,0:28:52.89,Default,,0000,0000,0000,,or germ layer formation,
Dialogue: 0,0:28:52.89,0:28:55.77,Default,,0000,0000,0000,,additional components\Nthat are not present here
Dialogue: 0,0:28:55.77,0:28:58.48,Default,,0000,0000,0000,,in this prototypic pre-initiation complex
Dialogue: 0,0:28:58.48,0:29:00.21,Default,,0000,0000,0000,,will come into play,
Dialogue: 0,0:29:00.21,0:29:03.42,Default,,0000,0000,0000,,and that's a subject\Nof subsequent lecture.
Dialogue: 0,0:29:03.42,0:29:06.36,Default,,0000,0000,0000,,But already you can tell that\Nthe transcriptional machinery
Dialogue: 0,0:29:06.36,0:29:08.67,Default,,0000,0000,0000,,is anything but simple.
Dialogue: 0,0:29:09.63,0:29:12.72,Default,,0000,0000,0000,,So, can we get a better\Nidea of what transcription
Dialogue: 0,0:29:12.72,0:29:16.44,Default,,0000,0000,0000,,might actually look like\Nand what's happening
Dialogue: 0,0:29:16.44,0:29:18.36,Default,,0000,0000,0000,,when a transcription process takes place?
Dialogue: 0,0:29:18.36,0:29:21.72,Default,,0000,0000,0000,,So, let me first of all say\Nthat I'm gonna finish my lecture
Dialogue: 0,0:29:21.72,0:29:24.45,Default,,0000,0000,0000,,now with a little cartoon,
Dialogue: 0,0:29:24.45,0:29:29.16,Default,,0000,0000,0000,,which is our attempt to imagine
Dialogue: 0,0:29:29.16,0:29:30.99,Default,,0000,0000,0000,,the events that take place
Dialogue: 0,0:29:30.99,0:29:33.00,Default,,0000,0000,0000,,when you form a pre-initiation complex,
Dialogue: 0,0:29:33.00,0:29:37.41,Default,,0000,0000,0000,,you bring regulatory proteins\Nto the activated gene
Dialogue: 0,0:29:37.41,0:29:40.02,Default,,0000,0000,0000,,and what happens during this process.
Dialogue: 0,0:29:40.02,0:29:43.53,Default,,0000,0000,0000,,Now, keep in mind that\Nthis is at this point
Dialogue: 0,0:29:43.53,0:29:47.70,Default,,0000,0000,0000,,mostly a cartoon that\Nis in our imagination
Dialogue: 0,0:29:47.70,0:29:52.70,Default,,0000,0000,0000,,and only parts or if any\Nof this is probably real,
Dialogue: 0,0:29:52.89,0:29:56.34,Default,,0000,0000,0000,,but it gives you a sense of the complexity
Dialogue: 0,0:29:56.34,0:29:58.80,Default,,0000,0000,0000,,of the transactions\Nthat have to take place
Dialogue: 0,0:29:58.80,0:30:02.04,Default,,0000,0000,0000,,just for one gene to\Ntranscribe and express itself.
Dialogue: 0,0:30:02.04,0:30:04.29,Default,,0000,0000,0000,,So, let me show you the movie,
Dialogue: 0,0:30:04.29,0:30:07.41,Default,,0000,0000,0000,,and then we'll finish\Njust by keeping in mind
Dialogue: 0,0:30:07.41,0:30:09.96,Default,,0000,0000,0000,,that there's much to be learned.
Dialogue: 0,0:30:09.96,0:30:13.29,Default,,0000,0000,0000,,And in my next lecture,\Nwe'll go into the selectivity
Dialogue: 0,0:30:13.29,0:30:16.56,Default,,0000,0000,0000,,of this process in specialized cell types.
Dialogue: 0,0:30:16.56,0:30:20.28,Default,,0000,0000,0000,,So, now let's see what\Nthis sort of this cartoon
Dialogue: 0,0:30:20.28,0:30:22.14,Default,,0000,0000,0000,,of transcription looks like.
Dialogue: 0,0:30:22.14,0:30:23.70,Default,,0000,0000,0000,,So, we start off with DNA
Dialogue: 0,0:30:23.70,0:30:26.79,Default,,0000,0000,0000,,with some preassembled TFIID molecule,
Dialogue: 0,0:30:26.79,0:30:28.80,Default,,0000,0000,0000,,and along comes this other green molecule,
Dialogue: 0,0:30:28.80,0:30:30.69,Default,,0000,0000,0000,,which is actually a co-factor,
Dialogue: 0,0:30:30.69,0:30:32.76,Default,,0000,0000,0000,,which then forms this very large complex
Dialogue: 0,0:30:32.76,0:30:34.17,Default,,0000,0000,0000,,with RNA polymerase.
Dialogue: 0,0:30:34.17,0:30:37.83,Default,,0000,0000,0000,,And then a distal\Nactivator protein came in
Dialogue: 0,0:30:37.83,0:30:39.27,Default,,0000,0000,0000,,and activated the process.
Dialogue: 0,0:30:39.27,0:30:44.27,Default,,0000,0000,0000,,And this molecule, this bluish\Nmolecule that's moved away
Dialogue: 0,0:30:44.61,0:30:48.06,Default,,0000,0000,0000,,from the complex is\Nactually the RNA polymerase.
Dialogue: 0,0:30:48.06,0:30:51.81,Default,,0000,0000,0000,,And that little yellow\Nsort of bead on a string
Dialogue: 0,0:30:51.81,0:30:53.64,Default,,0000,0000,0000,,is actually the RNA product.
Dialogue: 0,0:30:53.64,0:30:57.66,Default,,0000,0000,0000,,So, that gives you a sense of\Nthings have to happen quickly
Dialogue: 0,0:30:57.66,0:30:59.85,Default,,0000,0000,0000,,and yet it involves many, many molecules
Dialogue: 0,0:30:59.85,0:31:02.46,Default,,0000,0000,0000,,having to assemble and then disassemble
Dialogue: 0,0:31:02.46,0:31:04.17,Default,,0000,0000,0000,,to give you this reaction to happen.
Dialogue: 0,0:31:04.17,0:31:06.81,Default,,0000,0000,0000,,And in my next lecture,
Dialogue: 0,0:31:06.81,0:31:10.38,Default,,0000,0000,0000,,we'll go into more specific\Naspects of this reaction,
Dialogue: 0,0:31:10.38,0:31:13.47,Default,,0000,0000,0000,,and particularly during\Nembryonic development
Dialogue: 0,0:31:13.47,0:31:16.20,Default,,0000,0000,0000,,and tissue-specific gene expression.