| 00:01 | Welcome. This is the uh third module. We're talking about different aspects |
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| 00:07 | pro period uh genetics. And in 10, we're gonna cover this in |
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| 00:14 | parts, part one. The overall theme here in chapter 10 is gene |
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| 00:19 | in prokaryotes. So we're gonna start part one and shown by the learning |
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| 00:27 | here uh about gene con about how gene expression is controlled in |
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| 00:34 | OK. The different levels at which controlled and remember, this is all |
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| 00:38 | to the flow of information, right to RN A to protein and that |
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| 00:45 | of that process occurs at multiple steps the way that the level of |
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| 00:50 | the level of RN A at the of protein. Uh In addition, |
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| 00:56 | gonna be different terminology. We'll talk terms induction repression, uh corepressor |
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| 01:03 | uh de repression, et cetera that go over uh the role of transcription |
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| 01:08 | and expression. Uh And we'll use our, our uh examples for pro |
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| 01:14 | gene control and the lactose operon and tryptophan operon and how um uh control |
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| 01:22 | control mechanisms are used to um to gene expression. OK. So, |
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| 01:32 | let's talk a little bit of background . So when we talk about controlling |
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| 01:38 | expression, why is that uh OK. So let's look at it |
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| 01:43 | the term, the perspective of the cell itself. OK. So out |
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| 01:49 | the environment where are all the different of uh conditions it gonna be subjected |
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| 01:55 | ? Right. So temperature, of , ph oxygen levels, nutrient levels |
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| 02:01 | various types, uh Sodi concentrations, th all these and there and there |
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| 02:06 | there's many more, but these are of the more major ones that a |
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| 02:10 | must contend with on a uh minute minute basis really and, and fluctuations |
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| 02:17 | these. And so there has to some kind of response to the cell |
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| 02:22 | these different conditions and um obviously critical that's critical to its survival. And |
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| 02:31 | , so how does this go Well, typically it's gonna be a |
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| 02:34 | of uh so the external signal, it's temperature ph or multiple of |
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| 02:41 | that external signal must somehow be translated a, an internal function that the |
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| 02:49 | well then counteract that um um OK. So a sensor protein is |
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| 03:00 | where and so it's all of course chemical signals and proteins, how this |
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| 03:04 | . And so the um external signal sensor protein, it is trans duced |
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| 03:11 | an internal signal right through internal And of course, this is typically |
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| 03:18 | be through the through the production of sort of protein that will bring about |
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| 03:23 | effect. Ok. So of gene gene expression, OK. And |
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| 03:28 | typically, this will of course involve promoter. Uh We, we already |
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| 03:33 | that pro cario genes are mostly organized OPERON so that uh metabolic pathways and |
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| 03:39 | types can be uh expressed at the time or shut off as needed. |
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| 03:45 | so the uh not only does this this kind of uh example, I'm |
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| 03:52 | you here relate to gene expression being on. It could be as |
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| 03:56 | Gene expression being turned off. So signal signal can produce both kinds of |
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| 04:01 | depending on uh what it is and the needs are of the cell. |
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| 04:07 | , of course, and if expression or protection of a protein and that |
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| 04:11 | produce some kind of response, Whether it's to initiate metabolism, if |
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| 04:17 | a nutrient of that nutrient, uh to synthesize something if that's required uh |
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| 04:23 | to counteract some kind of negative effect some way. So all these |
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| 04:29 | are possible responses. OK. And , it could, yeah, soccer |
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| 04:35 | be rarely. Is it just just turning on one operon or one |
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| 04:43 | It's typically multiple genes, operon. some being turned on, some being |
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| 04:48 | off again, it all depends on the signal is. OK. So |
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| 04:53 | terms of controlling what's going on, this is where we look at the |
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| 04:58 | levels of, of the flow of . Right. So, starting with |
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| 05:03 | and so uh the soap might modify , right. Typically methylation, putting |
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| 05:08 | groups onto specific nucleotides can alter gene . Um rearrangements of DNA. Th |
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| 05:17 | phenomenon called phase variation, which we'll talk about particularly important in the context |
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| 05:23 | pathogenic microbes. Um So simply by the DNA code itself, uh that |
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| 05:32 | affect gene expression. So, of , we can go to RN A |
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| 05:38 | that, of course, we have transcription, right, the production of |
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| 05:43 | , of a messenger RN A from gene DNA. And so if we |
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| 05:49 | alter that somehow, so either allowing not allowing the RN A prelimerase to |
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| 05:54 | a transcript would be the level of control. Um Now, what can |
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| 06:00 | be confusing is that people mistake transcriptional for um a modification of the transcript |
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| 06:11 | . OK. Um Transcriptional control refers whether or not transcription will be |
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| 06:19 | So that's gonna affect the level of the functioning of the R ply, |
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| 06:24 | . So typically, those involve actions occur around the promoter operator region, |
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| 06:29 | to either allow or prevent transcription. what transcriptional control is. The post |
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| 06:35 | control uh comes can come after the is made. OK. And so |
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| 06:42 | stability of RN A is somehow altered in some way. Uh The |
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| 06:49 | in general pro Caro transcripts, messenger do not have a long lifetime. |
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| 06:57 | on the order of minutes. All . Um if the cell needs more |
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| 07:03 | for that particular gene or genes, more can be synthesized if it's |
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| 07:07 | So it's very tightly controlled. And , and the um and for that |
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| 07:14 | , it could because as long as transcript is hanging around the cell, |
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| 07:16 | will be translated. So remember that cells are efficient and it won't |
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| 07:21 | to, to transcribe an uh a indefinitely. There's gonna be a lifetime |
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| 07:28 | it um because it will only likely it for a certain period of time |
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| 07:33 | . OK. So again, you have to remember this, these processes |
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| 07:36 | all energy consuming. OK. Inscription . So, so it's not gonna |
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| 07:41 | to waste energy when it doesn't need be doing that. So that's why |
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| 07:44 | processes are all tightly controlled and can uh controlled at multiple levels, |
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| 07:53 | Translation, right. So uh translational , which does fall under the umbrella |
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| 07:59 | posttranscriptional control. OK. Uh Affecting of course means you're affecting the |
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| 08:08 | Is it going to be allowed to translate that transcript? OK. And |
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| 08:15 | different ways that can happen. Um translation of course now refers to has |
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| 08:22 | protein been made, right? And it has that can still be uh |
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| 08:28 | , altered, um because you get of the protein, practically stopping expression |
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| 08:34 | that gene. And so many times need to be activated once they're made |
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| 08:38 | that can be done through addition of groups is very common. Uh that |
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| 08:43 | also maybe inhibit a protein. So can affect proteins this way by modifying |
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| 08:48 | uh chemically through the addition of different of molecules to it. Or you |
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| 08:54 | degrade it. Proteins can be marked degradation. Uh proteins have a finite |
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| 08:59 | and can uh accumulate damage over So, uh it's necessary in some |
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| 09:05 | to get rid of those proteins before become due too uh detrimental to the |
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| 09:10 | . OK. So while while many, many genes are of course |
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| 09:15 | controlled in various ways through more and of these mechanisms just mentioned, there |
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| 09:21 | always a kind of a set group genes that are always expressed. |
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| 09:26 | And you might, you might consider the core genes we talked about before |
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| 09:32 | of those fall into this group of , they're always expressed. There's such |
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| 09:36 | critical um uh function for the cell they always need to be um |
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| 09:44 | Uh things like uh perhaps uh genes in in glycolysis and solar respiration. |
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| 09:50 | example, because cells cont continue need produce energy. Um So those are |
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| 09:55 | example of the types of, of that might always be expressed. |
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| 09:59 | So um so we call those Um Now, in terms of |
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| 10:07 | when we talk about in expression and there, of course, are uh |
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| 10:15 | genes and there are proteins involved in process as well. And so we |
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| 10:21 | categorize these as repressors and activators. there's terms that go with that called |
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| 10:27 | induction and called repressor de repression, ? And so these refer to um |
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| 10:33 | know, the gene being uh whether is occurring or being blocked. |
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| 10:37 | So it goes kind of both And so it's a generic model, |
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| 10:41 | see there where there's a regulatory protein interacting with a regulatory sequence. |
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| 10:49 | And then that brings about some sort effect on expression. OK. And |
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| 10:57 | we talk about induction and de So induction means expression is on, |
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| 11:02 | inducing, turning on expression. Um De repression. If something is |
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| 11:09 | , that means it's, it's being , it's being um turned down or |
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| 11:14 | off. OK. So if we it, it means we're taking away |
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| 11:20 | blockage or that limit. And so allowing it to, in this case |
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| 11:23 | expressed, right? So induction and are similar and that refers to the |
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| 11:30 | gene is being expressed gene or genes being expressed. And so here is |
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| 11:35 | example of induction. So an inducible we call it of which lactose is |
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| 11:43 | example of that, that we'll look um the in the uh inducible operon |
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| 11:50 | turned on expressed when an inducer is . OK. And so, and |
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| 11:57 | see that right here, OK, . And so, so we have |
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| 12:07 | states here, right? We have state where the um with pressure protein |
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| 12:16 | present, I pressure protein is gonna involved in, in altering expression. |
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| 12:22 | so the repressor protein can be in states, right, either it's |
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| 12:29 | right? So an active repressor is that uh will do its function, |
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| 12:35 | will repress expression if it's active. . And that's what you see there |
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| 12:40 | its active state, it's binding to regulatory sequence. OK. Right |
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| 12:46 | Food and green and in doing so gene is not being able to be |
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| 12:52 | . Now, we alleviate that expression inactivating the repressor as we see |
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| 12:57 | So, in the inactive state, see how they induce her showing green |
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| 13:03 | bound to that repressor producing an inactive . OK. So when that |
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| 13:12 | when the inducer binds the shape of repressor changes, such that it can |
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| 13:16 | longer bind the regulatory sequence and comes . OK? And now we're able |
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| 13:21 | express the gene. OK. So we look at a um at a |
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| 13:30 | repression system, a repressible operon, typically called, um we can't have |
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| 13:38 | involvement of a COVID pressure. So in this scenario, gene expression |
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| 13:45 | stopped when it's repressed. All if we look at it here, |
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| 13:50 | is our repressor protein. OK. here is the corepressor. So it |
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| 13:59 | a different role in this, in model here below the ligand does. |
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| 14:06 | it acts as a co oppressor in particular gene regulation mechanism. And when |
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| 14:11 | binds to the repressor, it, forms the active complex, right. |
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| 14:16 | that's the opposite of the inducible system see above right, uh inducer binding |
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| 14:22 | the repressor created an inactive repressor which expression in the de repression model, |
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| 14:30 | corepressor bound binds to the repressor, it. And so that active complex |
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| 14:37 | then block the expression. So kind two different ways. But but the |
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| 14:42 | line is so here we see uh right, inactive repressor. So when |
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| 14:51 | corepressor is not bound, right? you see it here is unbound repressor |
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| 14:58 | active. OK? And so this so we have active complex and |
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| 15:06 | So they're actually the opposite in terms how they work, the repression versus |
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| 15:12 | . But what the what the constant the constant is when, when we |
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| 15:18 | about an active repression, whether it's the induction model or the de repression |
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| 15:23 | , the active repressor means the same , it's just achieved in a different |
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| 15:29 | . Active repressor means the repressor can gene expression, right? That is |
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| 15:34 | . No matter what system we're talking . Similarly, an inactive repressor will |
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| 15:42 | a block expression. Gene expression occurs an inactive repressor is present. And |
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| 15:48 | true. For either scenario, it's what it takes to make an active |
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| 15:52 | and what it takes to make the repressor may completely different in those two |
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| 15:57 | as we saw. But the meaning those is the same. OK? |
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| 16:04 | that's what we'll see as we look the lactose operon and the Aryan opera |
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| 16:10 | how they're controlled. So, all right. So uh a word |
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| 16:19 | transcriptional activators, OK. So, activators, um as you might |
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| 16:28 | promote expression, right, very often can have the conditions that allow for |
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| 16:35 | to occur, like having an inactive present. But in many cases that |
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| 16:41 | is not enough, OK? You to in include uh the functioning of |
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| 16:47 | , of a, of an activator s to significantly increase transcription. And |
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| 16:55 | , you know, we'll see this the lac operon like how that |
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| 17:00 | So again, you can have what see here, a basal level of |
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| 17:07 | that generally is not at a level productive enough for the cell to |
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| 17:12 | achieve anything. OK? Um It only produce a few molecules worth of |
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| 17:19 | , right. So they get to get higher levels of expression. |
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| 17:22 | typically have to include the, the of an activator and that's what that |
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| 17:28 | . So there can be, as see uh with the glucose, with |
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| 17:32 | , I'm sorry, with the lactose that there can be a, a |
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| 17:39 | a repressor may be inactivated that will gene expression. But that in itself |
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| 17:44 | enough that we ha we have to the, the presence of an activator |
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| 17:48 | increase expression. And there's reasons for , that we'll, we'll talk |
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| 17:53 | So, and what it really boils to is and we talked about this |
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| 17:58 | little bit earlier previously in chapter 78 strong, which is weak promoters, |
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| 18:04 | , that high level expression is about R PLY molecule that binds the promoter |
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| 18:10 | then copies and makes the transcript to gene expression. That that is enhanced |
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| 18:16 | increasing the affinity, the binding of R PLY to the promoter. |
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| 18:23 | If that's really high, then that to more expression. OK. And |
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| 18:29 | really about, that's really what what is about. But by introducing an |
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| 18:33 | is to kind of increase the affinity that po for that promoter to increase |
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| 18:39 | . OK. That's really what it down to. OK. So uh |
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| 18:45 | look at the Black Opera. So um Black Opera as we see |
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| 18:53 | So remember the opera structure, We have a promoter and we have |
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| 18:56 | genes and a regulatory sequence that's can close by or can be far |
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| 19:03 | OK. So the laboratory sequence here the um a black eye gene and |
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| 19:14 | with pressure. OK. Now, la Opera itself contains these three structural |
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| 19:20 | , beta glacy lactose perm ase and black a called tho galactosidase trans |
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| 19:27 | OK? You don't need to worry that. OK? Because this, |
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| 19:35 | gene is, it's still really not what its function is because number |
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| 19:40 | this, this operon enables the bacterium possesses it to be able to utilize |
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| 19:46 | lactose is a sugar similar structure to that can be used as an energy |
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| 19:52 | . OK. Food source for energy having opera enables the cell to take |
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| 19:58 | in and to begin to metabolize OK. That lack a gene uh |
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| 20:05 | nothing to do with that process. An operon lacking that gene, a |
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| 20:13 | that has an opera lacking that gene still metabolize lactose. So that's why |
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| 20:19 | than knowing it's part of the lac . Other than that, you don't |
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| 20:23 | to worry about it because it doesn't function in this in, in |
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| 20:27 | lactose anyway. So let me So lactose operon is a canna bolic |
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| 20:34 | , right? We'll talk about metabolism the next, you know, but |
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| 20:37 | is how you basically how you take large organic molecules, food, |
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| 20:43 | And break those down and get energy them. So in this case, |
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| 20:47 | for lactose, OK? Um so in contrast, we talk about |
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| 20:54 | trip different operon uh which we'll get into in the next, in part |
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| 21:00 | pin operon is a anabolic operon. , it's about expressing the genes that |
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| 21:08 | for the synthesis of an amino OK. The black opera is completely |
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| 21:13 | . It's about expressing genes that are to metabolize a particular food source. |
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| 21:19 | ? And that goes away ways into why the control mechanism is the way |
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| 21:25 | is. OK. So now, of the first things to understand is |
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| 21:34 | there is always a a low level lack operan transcription occurring and by |
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| 21:42 | I mean very, very low. . So when the up run is |
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| 21:53 | repressed, OK. Um when the repressor is present and blocking transcription, |
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| 22:04 | that that binding B binding of protein DNA, it's typically not irreversible, |
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| 22:14 | always reversible. It comes on, comes off with a certain, there's |
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| 22:17 | certain equilibrium in which that happens. . Um Binding constant is a term |
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| 22:23 | for that. And so the binding an active repressor to the the the |
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| 22:32 | region of the opera, he's not co it's the comes on, it |
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| 22:39 | off, I mean, granted is on more than it's not obviously, |
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| 22:43 | there are short intervals of time where not bad. OK? And those |
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| 22:48 | opportunities to get some low level OK. Now, the very for |
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| 22:54 | where the repressor binds is what's called operator, right? We're familiar with |
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| 22:58 | operator structure. And um so back why is there a low level of |
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| 23:08 | ? OK. So let's look at components of the opera. So the |
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| 23:14 | ase the lac Y product is a , it's a perm. So perm |
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| 23:19 | is a membrane protein serves to bind and bring lactose into the |
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| 23:25 | OK? Once in the cell, would become metabolized, right? And |
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| 23:30 | two routes. So there's this enzyme bey the lac Z product. So |
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| 23:36 | low level uh on low levels of suppressant, it will implement in the |
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| 23:42 | , it will convert that to the lactose. Allo lactose is, is |
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| 23:47 | the inducer. It's the molecule that bind to the repressor and inactivate |
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| 23:53 | Ok. Also when, as lactose , the increase in the cell, |
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| 24:00 | the lac Z um brings about the part where it will then fall into |
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| 24:08 | glycolysis and cell respiration to produce So, lactose is a dissect or |
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| 24:13 | think it's cle into g lactose and . And that's through the lack of |
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| 24:16 | enzyme, glucose will then funnel into . G lactose will as well, |
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| 24:20 | it has to go through an extra . OK. So um so let's |
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| 24:27 | at why there has to be a level of expression of the black opera |
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| 24:33 | only way. So again, back the what I said before. So |
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| 24:37 | a scenario where we have the R plume race in orange, right? |
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| 24:41 | our lack of pressure is a right? It's bound to the operator |
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| 24:44 | where either P is promoter, O operator the. And so this is |
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| 24:49 | scenario where the oppressor is active, bound to the operator. OK. |
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| 24:57 | , if this were a binding, that no expression ever occurred, you |
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| 25:01 | never express these enzymes, obviously express genes for those enzymes. The only |
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| 25:10 | that this eco and E coli has a is an example of a bacterium |
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| 25:14 | can uh has lack up on the way that, that eco eo can |
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| 25:21 | see that lactose is out here if were present is if it had the |
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| 25:32 | the black Y sitting in the OK. So here's the la Y |
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| 25:38 | Z. So, so as I , you know, even in a |
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| 25:42 | where the, the um the pressure is active and bogging expression, it's |
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| 25:50 | a permanent binding, you know, of the time their pressure is bound |
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| 25:55 | the operator, right? But there's 0.001% or it's not right. And |
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| 26:01 | where plumbers can sneak in and, make a few molecules worth right |
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| 26:06 | of product. OK? And so when a little bit of lac Z |
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| 26:11 | Y can be made and that's essential the lac Y is what allows the |
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| 26:16 | coli to see if there's even any out there. OK. So that's |
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| 26:20 | there's always a super tiny, low of expression. And by low, |
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| 26:24 | mean, super low, we're talking maybe one or two lac Y permeates |
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| 26:29 | are produced. And so they sit the cell and that's the way |
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| 26:32 | for the cell to see if there's lactose out there. OK. So |
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| 26:37 | there is, then, so think Macquire is the lactose detector. That's |
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| 26:43 | I think about it, right? if it's out there maw will bind |
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| 26:48 | it, come into the cell and very quickly, right? So lac |
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| 26:54 | will serve to produce all lactose which bind to the repressor and then allow |
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| 27:02 | to occur. And if there's enough out there, or I should |
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| 27:08 | if there's a lot of lactose out very, very quickly will, will |
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| 27:13 | ramp up. So in, in , in this low level expression that |
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| 27:18 | talking about initially, again, we're about one or two molecules of, |
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| 27:24 | pro of gene product are made. it's very low. So it's not |
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| 27:27 | , a huge burden on the cell any means. But that level of |
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| 27:32 | which you would term basal level can ramp up 1000 fold, right? |
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| 27:38 | there's lots of lactose that comes the l lactose would be made and |
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| 27:44 | block uh you know, inactivate the . And you'll, like I |
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| 27:48 | you'll have 1000 fold increase in expression very quickly process that glutose lactose and |
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| 27:54 | utilize it. So here are the scenarios. OK. So again, |
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| 27:59 | lack repressor interacts with lack operator OK, to block expression. And |
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| 28:09 | , what happens is the repressor uh to both. So the, so |
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| 28:15 | lack uh the lack eye repressor gene an operator as does the lac lactose |
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| 28:23 | has an operator. And so when repressor binds, it kind of actually |
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| 28:26 | those both of those together. And effectively does not allow RN a pliers |
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| 28:31 | bind and you don't get any right? So absence of lactose |
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| 28:36 | presence of all lactose induction. And so, of course, because |
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| 28:42 | inducer lactose comes into the cell, of it's formed in the Allal Latos |
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| 28:47 | the inducer binds to the repressor and it. Right. So, repressor |
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| 28:53 | in that scenario on the left repressor on the scenario on the right. |
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| 28:59 | . So, uh um so that's it works. Now, there is |
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| 29:10 | another layer of control to this. . And that is with the, |
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| 29:17 | it's one thing to have the scenario see here. OK. On this |
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| 29:30 | , it's still low level expression because need to involve the presence of an |
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| 29:34 | . And that's what we're gonna see the next slide. OK. So |
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| 29:39 | is through the formation of a, active uh transcription, an active transcript |
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| 29:49 | complex. OK. So it involves molecule called cyclic MP and um |
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| 29:56 | a regulatory protein called CRP which um for cyclic A and P receptor |
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| 30:06 | OK. So in the active complex second AM PC RP protein bind to |
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| 30:17 | lack promoter, greatly increasing expression, ? Transcription. Excuse me. And |
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| 30:26 | that you see there um right here the C A MP levels, the |
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| 30:38 | receptor protein is more or less a of uh constant levels. But it's |
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| 30:43 | cyclic A MP molecule that can OK. So if there's plentiful cyclic |
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| 30:53 | MP around, then you're gonna form of active complex and you need to |
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| 30:56 | high level expression. So the key what is controlling the levels of cyclic |
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| 31:01 | MP because that's what influences whether you none low or high expression of that |
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| 31:10 | . And so we'll see here that influence is a, is a |
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| 31:14 | So glucose levels, so cellular cyclical P levels fluctuate uh depending on what |
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| 31:22 | sources energy level is. Ok. glucose has a heavy influence and that's |
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| 31:26 | glucose is the preferred carbohydrate source. glucose when it comes into a cell |
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| 31:35 | , glucose, six phosphate that goes into glycolysis. Ok. As you |
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| 31:43 | lactose requires some processing before it can glycolysis and then go into cell |
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| 31:51 | Um You have to cleave lactose. after you do that, you form |
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| 31:57 | , but then you have gla lactose has to be processed. So it's |
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| 32:00 | little bit, it's less efficient to than this glucose. So, glucose |
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| 32:04 | present if glucose and lactose are built , it's preferentially gonna use glucose because |
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| 32:09 | a more higher efficient way to, to utilize. It's high, it's |
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| 32:14 | more, it doesn't require more steps use. So, glucose, glucose |
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| 32:20 | , it can funnel right into the pathway and and be processed to produce |
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| 32:25 | . So glucose is first and then carbohydrates behind it. And it's through |
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| 32:31 | , the manipulation of cyclic E MP , how this occurs. So, |
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| 32:35 | there's high levels of glucose in the , there's a result in low levels |
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| 32:39 | cyclic A MP. OK. when there's low glucose levels, |
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| 32:44 | high levels of cyclic A MP. there are a number of um |
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| 32:49 | uh other carbohydrate operon like lactose that controlled the same way. OK. |
|
| 32:55 | , but glucose exerts its influence over of them. OK. And |
|
| 33:02 | um again, when um the active forms because cyclic A MP is present |
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| 33:08 | there's low glucose present or no then that opera will be active and |
|
| 33:14 | get the production in this case of la opera genes. So again, |
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| 33:20 | phenomenon of metabolite repression occurs when both and lactose are present, right? |
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| 33:27 | lactose is repressed, right? So is a the tab light. Uh |
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| 33:32 | is glucose actually. But you in the context of this, this |
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| 33:38 | glucose is, is repressing the expression these other contol lights, right? |
|
| 33:43 | so it's several sugars that fall into category besides lactose. OK. |
|
| 33:49 | so what happens when you do have of these together lactose, glucose and |
|
| 33:53 | in the medium? OK. here's an example of uh growth of |
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| 34:01 | coli where you have the glucose and present. OK. So glucose is |
|
| 34:06 | first and so you see the initial curve, OK. Then there's a |
|
| 34:13 | right here, right? And that course, is where gene expression is |
|
| 34:17 | switched over, right? We're expressing , we're shutting off the we're turning |
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| 34:21 | the, you know, we've accumulated C A MP that now we can |
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| 34:24 | on the expression of lactose genes. then once they're expressed, then very |
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| 34:30 | lactose gets utilized, right? So what we call dioxin growth or biphasic |
|
| 34:37 | if you will. OK. And mechanism of this, of how this |
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| 34:42 | is what's called inducer exclusion. So in a nutshell, the presence |
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| 34:47 | glucose uh actually blocks the transport of . OK. So it works like |
|
| 34:54 | . So you may recall this uh mentioned this in chapter three, I |
|
| 35:00 | on um transport mechanisms. And so is the uh um fossil transfer a |
|
| 35:08 | . So we use glucose and Manta examples of this. So here with |
|
| 35:13 | , uh the transporter is linked to subs that are phosphorated. OK. |
|
| 35:21 | so as glucose comes in at it will become phosphor the glucose six |
|
| 35:27 | and that goes right in to the . OK. That causes a cell |
|
| 35:34 | . And uh in that scenario, . This sub unit in the middle |
|
| 35:39 | two, OK. Is un Is it hands off that phosphate to |
|
| 35:47 | ? And so in that state that unit interacts with the lacy perm as |
|
| 35:52 | see there blocking expression, OK. of its present can't enter the |
|
| 36:00 | OK. So now if glucose is , yeah, then um these groups |
|
| 36:12 | phosphorated. OK? And in that , this is blocked, right? |
|
| 36:21 | can't interact with likewise. And so is out there, it'll come |
|
| 36:25 | OK. So in that scenario, on the right, where glucose is |
|
| 36:30 | , cyclic A and P levels will . OK. Lactose to lac peria |
|
| 36:35 | not interfered with uh and can come . OK? Because lac glucose is |
|
| 36:41 | absence. So lactose can come in very quickly. Um we have the |
|
| 36:49 | um activator complex formed, we get levels of expression and lactose can be |
|
| 36:54 | and processed for energy. OK. uh again called the, the uh |
|
| 37:01 | exclusion, right. Lactose being the exactly the inducer. It's all |
|
| 37:06 | but it's being excluded from the cell of the presence of glucose through this |
|
| 37:10 | . OK. Um So uh let's at this animation and these are available |
|
| 37:20 | you on the blackboard as well. look at um the lactose model |
|
| 37:28 | OK. So here is E coli this Black opera on. Yeah, |
|
| 37:33 | just go through this here. So uh the lac operon segment of the |
|
| 37:41 | and shown in uh blown up on insect. There's your lac Zy and |
|
| 37:48 | only focusing on those two. Uh your lac perm, right? That's |
|
| 37:52 | product. So lac Z is the gene product is be lactose like |
|
| 37:58 | the product is lactose perm like a that molecule you see there? But |
|
| 38:06 | focus on that. OK. Um then here's a scenario we have |
|
| 38:12 | right? So glucose present lactose of course, lac aprons repressed, |
|
| 38:18 | ? There's no lactose present at Uh So, um so here's a |
|
| 38:25 | where we see the lack I remember the regulatory gene producing the repressor protein |
|
| 38:32 | repressor and that will act on the , right? And you see there |
|
| 38:38 | , there's the lactose operon operator and lack eye operator in the presence of |
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| 38:46 | protein will serve to bring these together effectively not allowing transcription to occur. |
|
| 38:53 | . So there you have an active um that blocks expression. Yeah, |
|
| 39:04 | you have uh there there is a though usually it's bound in block |
|
| 39:13 | you can have, as I a, a small number of a |
|
| 39:18 | level of expression can occur when, this um pressure protein is not |
|
| 39:24 | So we get a little bit of . So remember, lactose perms are |
|
| 39:28 | detector, right? And so if is present, it can be detected |
|
| 39:34 | lactose perm ase which brings it in brings about um the cleavage of lactose |
|
| 39:42 | the two sugars monosaccharide as well as lol lactose, right? And lol |
|
| 39:47 | is actually the inducer. OK. it combined the repressor and in doing |
|
| 39:53 | , create an act, an inactive , right? So now we have |
|
| 39:57 | inactive repressor that can't bind and we expression, right. So in this |
|
| 40:02 | , then where we have glucose absent present, of course, we get |
|
| 40:08 | expression. OK. Um IOPS induced we have. And so you see |
|
| 40:17 | , the inclusion of the um activator , right? The cyclic A PC |
|
| 40:25 | . So when glucoses absent or very , you have high levels of soccer |
|
| 40:29 | P that will allow the formation of activator and produce high levels of |
|
| 40:36 | Now, glucose is present, all , we are suppressing the levels of |
|
| 40:41 | A MP as well as lactose is allowed to enter the cells. I |
|
| 40:45 | the the inducer exclusion model, And so we don't get um low |
|
| 40:53 | of C A MP, no aur . And we get this by basic |
|
| 40:57 | of growth. If both lactose and are present, glucose is less |
|
| 41:01 | then lactose. OK. So the here in the beginning where glucose was |
|
| 41:07 | is like a tab like repression, the expression of the lac oon. |
|
| 41:14 | . So that's that scenario. So we go to the trip operon, |
|
| 41:21 | . So just a couple of remember, the lactose operon is a |
|
| 41:24 | bolic operon, right? It involves the breakdown of the lactose sugar to |
|
| 41:31 | the goal of which is to get into like causes and cell respiration. |
|
| 41:35 | the cell can get energy right? . Tryptophan operon is the opposite is |
|
| 41:40 | it's a anabolic bio synthetic pathway. the the gene products of the trip |
|
| 41:46 | serve to, yeah are enzymes that to synthesize tryptophan, right? And |
|
| 41:53 | the 20 operon for each of the acids operate the same way. |
|
| 41:59 | And so there's five genes involved in hip hop run. And so |
|
| 42:05 | kind of review, right of, operon structure and bacteria, right. |
|
| 42:08 | yet the it it's an operon, structural genes are under the control of |
|
| 42:12 | promoter when transcription occurs as one big , right. Polycysts message right. |
|
| 42:20 | then that will be translated into the enzymes of the pathway charis mate is |
|
| 42:27 | the starting material. The raw material to synthesize tryptophan as it goes through |
|
| 42:31 | steps involving these enzymes and result as form krypto fan. OK. So |
|
| 42:40 | , anabolic pathway synthesis making stuff. . Yeah, repression, right. |
|
| 42:47 | there's a trip regulatory region, triple producing a trip repressor and that repressor |
|
| 42:55 | have two states of course, like a repressor, an active and inactive |
|
| 42:59 | . OK. The inactive state is where. So there. So the |
|
| 43:05 | opera involves the the inclusion of a that you see there, right? |
|
| 43:12 | the co oppressor is tryptophan itself. . So in the inactive state, |
|
| 43:20 | , the COVID pressure tryptophan is not to the um repression. OK. |
|
| 43:30 | call that form the apo repressor when trip corepressor is not bound to |
|
| 43:36 | So that form occurs when there's little no tryptophan present. So in that |
|
| 43:42 | , you will have the repression transcription occurring. OK. So again, |
|
| 43:50 | is kind of controlling its own So if crypt dorene is absent or |
|
| 43:55 | low, it's not really free to the trip repressor. So in that |
|
| 44:01 | , the repressor is inactive, And you keep, and you continue |
|
| 44:05 | get expression of the trip opera. right. Now, why is |
|
| 44:11 | OK. You, you have to of the role of crypt effect, |
|
| 44:22 | ? The pr is a critical amino , poly amino acids are critical, |
|
| 44:27 | course, because they're needed to produce . And of course, the points |
|
| 44:32 | proteins we're we're aware of right. are what do the functions of a |
|
| 44:36 | . So uh for every protein in cell there is gonna at least be |
|
| 44:44 | tryptophan, right? So it's gonna a critical function to keep crypto |
|
| 44:48 | Yeah, uh available, right? is needed. So it goes to |
|
| 44:54 | is when are mean life is needed in there's the need for them the |
|
| 45:01 | . Well, it's gonna be when are actively growing, right? Cells |
|
| 45:05 | actively growing. Cells are multiplying. we saw from the discussion on bacterial |
|
| 45:11 | that you know, lots of stuff needed and cells are dividing and growing |
|
| 45:15 | ? And chief among them is So the need for different amino acids |
|
| 45:20 | gonna be very high during periods of growth. OK. And um so |
|
| 45:26 | that, in that scenario, tryptophan likely going to be used as fast |
|
| 45:32 | being made, particularly in an actively culture. OK? Because they have |
|
| 45:37 | keep up with protein production and you to keep making crypto plan, it |
|
| 45:39 | getting funneled into making proteins. So amount of actual free tryptophan in a |
|
| 45:45 | in those periods is likely very which is why it keeps synthesizing more |
|
| 45:50 | and and the other amino acids is keep up with production of new cells |
|
| 45:55 | , as the cultures grow. um so that's, that's kind of |
|
| 46:01 | it in a way self regulates OK? Now, when tryptophan |
|
| 46:07 | right. So tryptophan is accumulating and now free to bind the repressor. |
|
| 46:13 | get it becomes active. OK? um in that scenario, then the |
|
| 46:28 | oppressor forms, right? So the pressure binds, it defines the core |
|
| 46:32 | , it binds and the um in form, it binds to the operator |
|
| 46:39 | the tryptophan opera and blocks expression, ? As you see there. So |
|
| 46:46 | of this in terms, think of in terms of of logic. |
|
| 46:51 | If um the cell is actively OK, there's a high demand for |
|
| 46:58 | acids, including tryptophan crypto will be and used likely very rapidly if |
|
| 47:06 | if the culture is growing quickly. and that makes sense, right? |
|
| 47:10 | you wanna keep up with demand. but when that culture begins to slow |
|
| 47:17 | , right, for whatever reason, it runs out of nutrients, |
|
| 47:21 | what have you right? Then all a sudden now you're, you're, |
|
| 47:25 | got this store of tryptophan that's not nowhere, it's not being used. |
|
| 47:30 | ? So it makes no sense on , on behalf of the cell to |
|
| 47:33 | synthesizing tryptophan if there's no demand, ? Again, remember always remember |
|
| 47:40 | you know, the transcription translation process try defense is all, there is |
|
| 47:43 | stuff that takes energy, right? , so it's gonna be efficient. |
|
| 47:47 | don't do it if it's not And so that's why what crypto begins |
|
| 47:53 | accumulate the soul goes, OK. stop. We don't, we don't |
|
| 47:57 | the same demand. Let's not keep energy. And so we're gonna block |
|
| 48:02 | . So that's, that's kind of , the perspective here. So if |
|
| 48:06 | look at it in terms of think it, in terms of relative importance |
|
| 48:10 | the LAC operon and the trip opera the South. OK. Clearly for |
|
| 48:16 | coli or in the bacterial cell, , if it's gonna choose between which |
|
| 48:22 | can I do without, it can't without tryptophan operon because it's, it's |
|
| 48:28 | only way for it to make that acid to put into its proteins. |
|
| 48:32 | can likely do without lac the lactose . Um There's lots of plenty of |
|
| 48:38 | so that don't have Black Opera um there's so many other ways uh so |
|
| 48:44 | other substrates that, that the E , for example, can use as |
|
| 48:47 | food source, but there's only one to make tryptophan and that's through that |
|
| 48:52 | . So it's got kind of goes why the the control logic is the |
|
| 48:56 | it is, right. Um Lac is not really fully expressed unless lactose |
|
| 49:01 | present in the environment, right, food source. But there's many other |
|
| 49:05 | choices to, to to choose from . But for the truth to find |
|
| 49:11 | similar to other bio synthetic o that mass pathways tryptophan, it it's for |
|
| 49:21 | production of that particular component. So thehan is produced as a result of |
|
| 49:26 | expression of the opera. So let's tryptophan is really important. So let's |
|
| 49:31 | kind of let it determine what right? Whether we get more expression |
|
| 49:37 | , or or uh less expression. . And uh it, it just |
|
| 49:43 | goes to the relative importance of both the opera of the cell, |
|
| 49:47 | ? Tryptophan is super critical, So we kind of let tryptophan levels |
|
| 49:52 | what happens that's more efficient for the . OK. So um uh |
|
| 49:59 | and it's, and it's a right? It's not, it's not |
|
| 50:03 | ever all on or all off. usually somewhere in the spectrum, |
|
| 50:07 | It all fluctuates depending on the needs the cell which typically loads of tryptophan |
|
| 50:12 | through, you know, what are um uh uh what's the growth state |
|
| 50:18 | the cell, right? Is it rapidly? Is it not? And |
|
| 50:20 | that's gonna determine the needs for the amino acids. OK. So kind |
|
| 50:25 | let the amino acids themselves kind of and that's gonna be a much more |
|
| 50:29 | way. So it kind of goes to some of the logic behind |
|
| 50:32 | . Um the uh and so what gonna do is uh this concludes to |
|
| 50:39 | one. So we'll expand on the control because it goes beyond as with |
|
| 50:45 | with lactose, OPERON, there's, was another other layer of control, |
|
| 50:50 | , the presence of glucose. So with tryptophan and Operon, there's another |
|
| 50:56 | of control. In addition. I so the couple another thing to |
|
| 51:06 | is that the um both the crypto , the control mechanisms we're looking at |
|
| 51:11 | for both the LAC and crypto an or what we call transcriptional control. |
|
| 51:15 | control are very common mechanism of control , in bacterial and arch operon. |
|
| 51:22 | . And so remember that transcriptional control all about, are we gonna allow |
|
| 51:26 | to occur or to not to OK. And again, a very |
|
| 51:30 | mechanism in in the pro cario OK. So let's look at a |
|
| 51:35 | bit of a summary here or question . So we answered that one. |
|
| 51:45 | um so let's look at um this here. Yeah. OK. So |
|
| 51:57 | question we have an E Coli which uh has both the LAC opera and |
|
| 52:03 | trip opera. And that's what LAC Trip plus refers to. I should |
|
| 52:07 | TRP is a shorthand for Tryptophan I'm sure you figured that out. |
|
| 52:13 | Trip is kind of how you say uh regardless. So, e coli |
|
| 52:17 | grown then in minimal medium, I , remember that, re remember what |
|
| 52:21 | means um containing both lactose and right? No glucose is present. |
|
| 52:30 | . Uh So again, lactose and tryptophan both present glucose is not all |
|
| 52:35 | . So what can you say about cells with respect to lack and trip |
|
| 52:39 | respectively? OK. So in terms uh in each scenario is the is |
|
| 52:45 | OPERON expressed. So if they lack expressed in this scenario, OK. |
|
| 52:54 | The um answer of course is right? Because there's no glucose |
|
| 53:01 | Number one lactose is present first and . All right. Uh And then |
|
| 53:06 | no glucose presence. So that tells a that uh well, we'll go |
|
| 53:11 | the whole question. I'm gonna give much away yet. But yes uh |
|
| 53:16 | um and I get this backwards. Let's just go through. OK. |
|
| 53:22 | yes, like opera and yes is , right? And um again, |
|
| 53:28 | is present, glucose is absent. ? The tryptophan opera. OK. |
|
| 53:34 | um so remember that tryptophan is in medium, right? So why would |
|
| 53:39 | would the E coli need to express trip opera on it's already being supplied |
|
| 53:45 | , right? So tryptophan comes in the cell. Uh what's not |
|
| 53:50 | It'll still be there and it can can block expression by creating, acting |
|
| 53:54 | the corepressor and activating the repressor So for that reason the burn will |
|
| 54:01 | be expressed, it doesn't need right? It's being supplied, cry |
|
| 54:03 | fan. Why make it if you're handed to it, it's being handed |
|
| 54:07 | you. Um What's the state of repressor? Right? So uh so |
|
| 54:11 | that the definition is the same regardless the opera, but it's one of |
|
| 54:15 | condition, the conditions can be different bring it about. OK. So |
|
| 54:19 | la opera is the uh repressor active inactive while it's being expressed, |
|
| 54:24 | So it's gonna be inactive, The other scenario, trip back uh |
|
| 54:29 | uh repressor is active, right? of blocking expression, right? The |
|
| 54:33 | already present in the medium is acting the corepressor to form the active repressor |
|
| 54:39 | . OK. Psychic A MP right? You didn't see it |
|
| 54:43 | You used to could tell that in presence of lactose and no glucose |
|
| 54:48 | That's the key that no glucose will to high levels of psychic A |
|
| 54:53 | And so that promotes formation of the A MP activator complex. That question |
|
| 54:58 | not applicable to the trip opera because not controlled by C A MP |
|
| 55:03 | OK. OK. So um so are kind of things as you go |
|
| 55:09 | this, you should be able to and contrast the opera similarities, |
|
| 55:16 | active repressor, an active repressor, know those kind of questions. |
|
| 55:21 | When is the cell going to be these opera at high levels, when |
|
| 55:26 | it not et cetera? So you be comfortable with that and look at |
|
| 55:30 | animations, right? We'll look at try to trippy opera animation in the |
|
| 55:35 | module. OK. So these are things we covered. So, um |
|
| 55:40 | know, kind of the overall, what gene expression is and why it's |
|
| 55:44 | um controlling gene expression through different Uh You should be familiar with the |
|
| 55:49 | of control uh level that level of versus transcriptional versus posttranscriptional, which can |
|
| 55:56 | translational and then post um translational which of course is the level of |
|
| 56:02 | protein. So you should be familiar that terminology. Uh And then of |
|
| 56:07 | , terminology as we talked about in to, to opera and the |
|
| 56:11 | right, repressors, inducers, co , activators, et cetera. |
|
| 56:17 | And then we went through the lactose . So you should understand that as |
|
| 56:22 | as the tryptophan operon. Although we a part of that still to do |
|
| 56:26 | we'll pick up next time. And so remember with the Tryptophan |
|
| 56:30 | we have the corepressor repressor uh uh . OK. All right. So |
|
| 56:37 | time will be part two, we'll finish off the tryptophan operon as |
|
| 56:41 | as go into some other control OK. Thanks |
|
