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00:02 Thanks. So let me just uh my screen and um we get

00:10 So, uh as before, uh me um put this in there,

00:18 uh section ID, just in case don't see it, put the clicker

00:27 oh four. OK. 4996841. . Um All right. So,

00:41 as you did before, if you questions, I'll periodically will um I'll

00:46 over at the chat section if you questions. Uh You can also just

00:50 uh unmute, unmute yourself and, ask a question. That's fine

00:55 So, um I, I'm I don't see your screen.

00:59 I haven't shared it yet. Uh So uh let me do that

01:06 . Um Three. OK. Yeah. And if you are mute

01:21 microphone, if you're out there so um you know, there you

01:25 OK. Uh Let me get a here. OK. So uh recall

01:36 I sent out the email uh regarding the classroom. So, uh because

01:45 in the Agnes Arnold Auditorium, uh won't be subjected to what those that

01:52 classes in Agnes part of the hall be. So we are just gonna

01:56 normal lab class in class sessions starting . Right. So from Monday to

02:03 rest of the semester, we're back as usual. Ok, so just

02:08 up in class, uh Tuesday, sorry, next Tuesday. So show

02:13 in class next Tuesday as you normally and we carry on. So no

02:19 remote uh classes like we're doing this . Ok. So again, just

02:25 to class next Tuesday right over in Ane Arnold Auditorium. Um What

02:31 Ok. So today we're continuing unit . So chapter seven and eight.

02:35 , so also kind of like with 21 22 we covered last time chapter

02:41 . Now I really pay attention to . Uh pay attention to this,

02:45 ? The what's gonna be covered because don't cover hardly, definitely not in

02:51 entirety though I covered seven and So I really do do focus on

02:55 what's here. OK. In terms the specifics of those two chapters,

02:59 . Uh I don't feel the need go into stuff you've had before in

03:04 detail. This is more kind uh my approach here is uh the

03:10 that are specific to pro Caros in the context of gene expression.

03:15 . So again, just kind of sure you, um stick to what's

03:19 and, and certainly don't, you need to know those, these chapters

03:22 their entirety. OK. Um All . The uh so of course exam

03:29 begins tomorrow uh through Saturday, depending when you're signed up for.

03:36 what else? Uh Yeah, I these are flip class. I'll have

03:40 number of questions here. But, , you know, uh and we'll

03:45 through some things and so I, usually this is kind of,

03:49 so today and next week, uh then the week after is kind of

03:55 uh an overview of bacterial genetics if will. OK. So today we

04:00 of start with um a bit of review really because uh some people are

04:06 , it may have been a while you've, you've had this material.

04:10 just, it's important to go over of the basics of, of genes

04:17 , and gene expression. Uh for of this, it'll be uh more

04:22 a review than others. Uh it may kind of just be,

04:25 a while since you've got, we've through that, you've gone through

04:28 So it's kind of just the first of this is kind of just the

04:31 getting you back into the thinking again how the whole process works in terms

04:35 gene expression. OK. And uh important to know that uh you

04:41 one because you're bio, bio right? Your bio majors for the

04:45 part, and it's something you just know as a bio major. But

04:50 us, you know, it's as get in the gene regulation coming up

04:54 week, if you don't know how process works, then how are you

04:58 really understand how it's controlled because they go hand to hand, right.

05:03 , so anyway, so we'll do little bit of a review here in

05:06 beginning with questions and, and Uh So if you have questions,

05:09 me know. Um other than so there will be a weekly quiz

05:13 week. Uh Yes, I know have an exam but the weekly quiz

05:17 really, it's not that there's maybe questions, just do it on

05:21 It's not, it's not gonna be big a deal. Ok? Um

05:25 no smart work due for another not the third of April. So don't

05:30 to worry about that. Um All . So as I mentioned, stick

05:34 this in terms of um what's gonna covered. And so we'll start with

05:41 question that pretty much will tell me you kind of know they see this

05:47 or not. It's one that uh is a 5050 response. So let's

05:52 at the process of transcription and translation carried out in a test tube.

06:00 . Two way test tube is So we have components from three different

06:05 . OK. So from a we're dumping in M R N A

06:10 R N A s and rhizomes from fish. We're dumping in, dating

06:15 a zebra. We're dumping in R plume in any other necessary enzymes,

06:24 and amino acids. So the question oops question is assume some new protein

06:36 made in the test tube. there will be the proteins of which

06:43 or animals will be expressed. So those are your choices. So

06:49 a look. Let me open the here. So uh pause it for

06:56 second. So uh so again, is uh this, this is

07:08 this can be done. You can the so called cell free extract.

07:11 you basically break up in the cells you can uh add the different components

07:17 of the process. And uh I'm sure if this exact experiment has been

07:24 , but some, some types like because it's, this process is a

07:29 translation that's universal, right? All based life uh carries it out pretty

07:37 the same way. All right, count down. OK. Number

08:08 Yeah. So that's kind of what thought. Um D and E so

08:15 pretty much um well, number So it's all, it's all based

08:22 this, of course. Right. here. Central dog. Right.

08:27 . See, we started learning that maybe as early as junior high high

08:32 , for sure, I'm guessing. nonetheless, um DNA R N A

08:36 , right? How the information flows all lives. OK. So,

08:44 DNA, right. Certainly. Uh proteins of the fish are certainly gonna

08:49 in there. OK? Because DNA , is the template, right?

08:54 But remember that M R N A right transcripts. So DNA to R

09:01 A, right? So I uh both the hippo and fish proteins are

09:07 here. OK? Because DNA is template, right? M R N

09:11 is a DNA version of the temp . OK? So even with the

09:15 DNA, because you have all the , the DNA will go to R

09:19 A to proteins. And the M N A is kind of by bypassing

09:23 , the DNA stuff and going from R N A to protein.

09:25 hippo and fish are gonna be the that are represented you. OK?

09:32 The zebra is not, there's no of template coming from the zebra.

09:37 ? So that's why it's not one part of this. OK. So

09:40 look at another question. OK. kind of relates to how you talk

09:47 genetics, I guess. So this uh for a certain bacterium. It

09:51 been found in the region of the designated X. OK. Uh comprises

09:58 specific protein coding sequence of DNA OK. Um The sequence, this

10:09 X can be converted into protein only the cells are grown on galactose.

10:18 a sole carbon source, galactose is type of sugar. OK. So

10:23 growing on the lactose, the, X uh X sequence is then converted

10:30 a protein when grown on the OK? Which is the following statements

10:36 these is true regarding the information about bacteria. So the two above sentences

10:43 of these is true. OK. So the X phenotype is revealed is

10:54 when cells are grown on glucose as carbon source. B the X sequence

11:01 a protein. See the conversion of DNA sequence into a protein starts with

11:10 binding to the X sequence of The X sequence is A K E

11:20 to the are all true statements. these sentences 1 and 2 above and then click

11:29 things, right? And don't make assumptions, just answer what you're giving

11:47 . I have one more question and will do some explanation here. Oh

12:03 I look at, there's any questions up up there? OK. Can

12:13 here? I assume, yeah, assuming everybody can hear me. I

12:17 have heard some heard by now. . OK. All right. Let's

12:35 down quick here. 15. All . X is a correct.

12:58 it, so obviously you'll answer So in any case. So um

13:04 X phenotype, so it says you to grow on galactose to see

13:09 All right, to see the phenotype sequence is a protein. It's a

13:13 sequence of A nucleotides, right? of course makes it A G uh

13:19 conversion, the sequence starts with um converting it into a transcript, not

13:26 his own binding. OK. So is of course A G. So

13:30 do one more, just kind of relating to the terms genotype and

13:36 OK, which can sometimes be miss , but uh just quickly go do

13:56 . And again, I realize this be kind of basic for some of

13:59 all, but some of you may have seen this in a while.

14:02 it's worth um worth a shot worth , rehashing it a little bit.

14:12 OK, so let's go what we here. So C and E are

14:22 . OK. So which is So C is definitely true. The

14:28 represents the expression of the, of . That's correct because again, it's

14:36 genotype. The phenotype take it as DNA to protein. Uh The

14:43 M type is always constant on a type may not be, that's true

14:50 well. OK. Um They're not interchangeably. So B and C are

14:57 , right? So two of A and C are true, which means

14:59 is true. So, um so and B and C are the two

15:04 ? OK. So A and genotype , let's just look at this real

15:11 here, right? So the phenotype from functioning of proteins, right?

15:17 again, in the DNA R N protein genotype is the DNA and pheno

15:23 is, is the protein is the . Typically, it's, it's expressed

15:29 um phenotype is often defined as the features an organism has, right?

15:37 so that translates into the um the uh proteins working at that particular time

15:50 produce that particular phenotype. Ok. here's an E coli, right?

15:56 E coli can ferment this sugar And there's a test that shows that

16:02 when it becomes yellow, acid results lactose. Fermentation. Yellow is positive

16:07 growing on lactose, bronze, Something that lactose negative would not,

16:11 it would not be able to convert and it would remain as a neutral

16:16 solution. And so um that's a , right? You can obviously see

16:22 right, a change in the growth uh based on the organ and being

16:26 to use that sugar. OK. the plate is another way to express

16:31 as well. So you can see the lactose phenotype is shown by the

16:37 pinkish colonies. OK? Um So now this statement about genotype is always

16:45 . I mean genotype is the the DNA is always there,

16:48 It's what you um prior to your dividing the DNA duplicates and those that

16:55 is passed on to daughter cells, cetera. Um this molecular inheritance as

17:00 all know. So it is a right? You always have it.

17:04 um but the phenotype can change uh on what's being expressed. And

17:10 and that's where gene regulation comes into , right? Controlling which genes are

17:18 expressed. OK? We will learn there are some genes that are pretty

17:22 expressed all the time. These are be things you might, you might

17:27 critical function type genes that always need be pretty much on all the

17:31 Things like genes involving glycolysis and right? Um Some are not always

17:40 , need to be expressed. There genes you haven't expressed since you

17:45 you know, 10 days old, ? And a developing uh develop from

17:50 to a developing fetus, right? lots of genes were being expressed back

17:55 and aren't being expressed now because you need them. You're a fully uh

18:00 adult, right? You're not in fetal stage anymore. So what genes

18:04 on or off will determine which phenotypes being expressed? OK. And that

18:10 , especially for cells that can change rapidly depending on environmental conditions of different

18:20 , nutrient availability. PH changes 02 levels, kinds of things, right?

18:27 can influence um what particular genes need be on or off. OK.

18:33 we'll explore more of that later but week, uh but that's why,

18:37 know, the G N type is , you know, may not be

18:41 . And so the certainly the right? So the genotype um is

18:48 into a phenotype through the mechanism of translation. OK. Um So the

18:58 so here is another different way to at. So especially for you a

19:02 , you know, you're doing this project, although you're not using this

19:05 , which is actually a, it's kind of a, a multi test

19:10 in one kind of uh uh cassette uh each compartment you see there,

19:16 only circled one, the urea or . Um the others are different biochemical

19:22 , right? You're doing these in lab with different test tubes,

19:25 This is just a different form and all the little compartments are inoculated and

19:32 then you wait to see what kind results you get, right? And

19:36 are typically always color based where a , if it has a metabolic

19:42 it will result in the P H typically. And you see the result

19:46 a color change, right? So only focusing on urea, OK.

19:50 urea uh test which uh if it's of using it will turn that compartment

19:59 . OK. And that indicates positive . OK. Um The, so

20:06 does that mean? Right. That's bit. So we can,

20:09 that's observable, we can see right? And that's another thing is

20:12 phenotypes may not always, certainly be , maybe they be they may be

20:19 . OK. Like for example, of all the metabolic reactions going on

20:22 your body, right? Those are phenotypes. Um but you're not not

20:29 visible to the naked eye, Uh If you can use lactose,

20:33 not gonna turn yellow, right? that's, you're, you're, you

20:36 , you're having a P change and have a P in the car.

20:39 ridiculous. But, but the point that not necessarily everything is absorbed with

20:43 naked eye, but you could measure different metabolic activities with um different types

20:48 tests, right? That's when you a physical, you get a blood

20:52 and those numbers represent your different phenotypes what their, what their levels

20:57 So, one way to look at anyway. So, back to

21:01 So here is a, a It's, it's positive for hydrolysis,

21:06 called. So what does that actually at the molecular level? Well,

21:11 is what the enzyme does. So enzyme is what produces this positive result

21:17 it hasn't, right, urea is down into uh decoupage and ammonia forms

21:23 that's becomes basic cos the color So then of course, ura enzyme

21:30 from a gene, right, the genotype. And so that uri a

21:36 , if in order to, to that enzyme, that protein, we

21:41 to go through the expression of that transcription, translation, transcription R N

21:46 polymerase copying that gene that DNA into R N A form. OK.

21:53 R N A messenger R N A . Same thing. OK. Then

22:00 the next part, excuse me, transfer R N A S. These

22:05 about the translation of that transcript into actual protein. Yeah. And then

22:13 will fold, typically folding is involved protein like peptide chain or changes depending

22:18 how big it is. And then enzyme protein folds into a shape that

22:23 becomes an active enzyme that can participate this reaction. OK. So a

22:28 of things, um uh is we're begin to focus more and more on

22:35 that for, for simplifications sake, just showing you this way. Uh

22:41 reality, you can form lots of . OK? All those transcripts will

22:47 lots of protein, right? So a cell, generally 11 transcript producing

22:56 protein is not gonna be enough, not meaningful. So it typically you're

23:00 produce lots of these things, That's where control comes in because you

23:04 to produce them for a period of , you need them, but you

23:07 want to rapidly shut it all off well. OK? And that's the

23:11 of regulation. OK. So, but regulation occurs at multiple levels.

23:18 you can see the multiple levels right? We've got DNA at the

23:23 of DNA, right? Um The we have the uh at the level

23:33 R N A, right, both transcription translation uh at the level of

23:37 protein and all three of these, , all three levels can you control

23:43 ? OK. I will see that we go through this unit.

23:47 So control is as equally as if not more. So the actual

23:52 of producing proteins because one thing that mentioned time and again, that you

23:59 get from these diagrams of whether it's replication or whether it's protein synthesis,

24:07 energy expenditure required for those processes. ? Remember if you're building a

24:13 you're that's analyst that takes energy, ? And so in a highly competitive

24:21 that these microbes are in, you want to waste energy, right?

24:26 that will put you at a So always being efficient is a big

24:32 and controlling your gene expression is a part of that. OK. So

24:39 so just continue on with this. kind of getting to more, more

24:43 the molecular level here. So your terms, you should know,

24:47 Transcription, translation and what those right? And again, I'm not

24:52 for the super detail here. Um You've had that before, I'm

24:56 uh I'm not gonna go into the workings of a, of a rhizome

25:00 , and all and all the various and stages. OK? It's more

25:04 of over them. OK? Um if you see transcription, you

25:10 what's the danger for that on a that produces an M R N

25:14 a messenger R N A, a , right? That's all part of

25:18 . So if you use an the the DNA is the constant,

25:23 ? So it's like the the book reserve in the library, OK?

25:28 can't take it home with you, ? So, but it's always gonna

25:32 there. And if you want some from that book, the DNA,

25:37 gotta make copies of it, So you take, you copy whatever

25:40 you want a xerox machine, that's transcription process, right? The transcription

25:47 . So you make copies, And those copies can be used.

25:50 so that's, that's in basic what's going on here as I'm sure

25:54 probably know. So the, with that transcript then um you,

26:02 that's the, that, that's called the working copy of DNA if you

26:06 , OK. So that, because can always make, you can always

26:09 more M R N A S Of A G always, you can

26:14 that as long as you're alive. um but the, the DNA is

26:18 permanent thing, right? So R A is come and go DNA is

26:22 . And um uh so with those , then you translate, that's what

26:27 the functional proteins, right? The thing to remember is that although most

26:32 , right? So the gene is core unit in DNA, right?

26:36 contains a sequence to produce a OK? That gene um uh

26:45 and most genes are just that protein genes, but there are a number

26:52 some that are not protein coding OK? Although they're so-called genes just

26:57 the end product of the gene is R N A, not a

27:01 And those are things like Rizo R A molecules transfer R N A

27:07 So there are genes for those and genes, the end product is simply

27:12 R N A, not a So just you know, remember that

27:16 not, not all genes are protein . Some are simply R N A

27:20 . OK. So uh so the thing to remember here is the

27:25 in right, there's no nuclear So polyribosome poly zone formation can

27:31 Transcription translation can occur virtually at the time. OK? Because there's no

27:37 of the process like there is in carry on. OK. So um

27:43 means you get lots of protein sens quickly. OK. So uh so

27:49 main things, right? So we ribosome, there's a ribosome binding

27:53 So the transcript, you see there a specific sequence and so ribosomes read

27:59 you will translate five prime to three . So you see that ribosome is

28:05 well on its way in in the of translation, it has bound the

28:09 binding site and then it up. remember there's punctuation in a, in

28:15 transcript. It's how it's read, how it becomes translated, right?

28:19 you have what are called um start or initiator codons, right? That

28:25 the beginning of the sentence if you uh then the co dots,

28:30 the three base nucleus ties that come after the initial codon, right.

28:37 you each codon has three bases, ? So you, you read it

28:40 that fashion. And so um and, and as you do,

28:45 produce a protein like peptide. So have the parts right, the ribosome

28:49 it brings it all together, it transcript, it provides a site for

28:54 sensors to occur. Uh or T N A S T R N A

28:58 come in and they are bringing a acid with them, right? That

29:06 to a particular codon. OK. I'll elaborate that in a second.

29:12 back to the sense, anti So we mentioned this in the context

29:16 viruses, right? And R N viruses. And so remembering that,

29:22 and so you see mark there on , the sense and anti sense strand

29:26 minus applies as well, right? so let's take a closer look at

29:32 same sequence you're seeing there here. . So the same sequence. And

29:40 uh so again, the, the way you talk about nucleic

29:45 which is a five prime end and three prime end. OK. And

29:50 uh so here's the complementary strands the sense and one is an anti

29:54 . OK. So the sense is a plus and the sense is a

29:59 . OK. So remember when you , you copy a minus, it

30:04 a plus. And as we saw the contact the virus, as you

30:08 plus, you, you, you a minus, you copy it into

30:11 minus strand. So regardless, so point here is that when we

30:16 when we're doing transcription, OK, DNA we are making a complimentary copy

30:23 the anti sense strand. OK. this one right here. OK.

30:33 of course, it's a minus right? So, because it is

30:37 copy with make up, that is plus strain. OK. And so

30:42 so, but it's an R N form, it's in the form of

30:45 N A, right? So remember no uh diamine in R N

30:50 they get replaced with Euro cells as see here. So when you compare

30:54 M R N A to the, the sense strand, they're identical,

30:59 ? Except for where you see a , right? So, so the

31:03 thymine there's a year or so. ? But except for that, they're

31:10 , right? And that's, that's you want because you're trying the,

31:13 , the, the sense strain of contains the information to make that

31:18 And we make that we get that the R N A form because we're

31:22 the antis senses or minus strain of . OK? And so now with

31:28 M R N A, we can . OK. So remember that's where

31:34 gene code table comes in, So uh my crude way to draw

31:40 T R N A. Yeah is like this. OK. So you

31:51 to kind of do it like this here's an amino acid A a amino

31:58 , OK? And then down here we call the anti Cota,

32:06 So the anti codon matches up with code, I can draw it

32:12 This and OK, so they match with the coat on and so in

32:23 so, right. A U G ? Match has a meth,

32:27 So the T R N A for would have the complimentary base to a

32:34 G, right? And then bring with it and it would with the

32:38 T R N A bringing the appropriate acid that corresponds to the code dot

32:43 . So, uh so that's how read a transcript translated rather to produce

32:49 approach. OK. So, you , that's the basics of transcription and

32:57 . OK. Um Let's uh Is I see a couple of people

33:09 able to hear, is anybody else to hear me? OK. All

33:19 , good. All right. All . Then it might be an

33:22 There's a couple of people, it be an issue with the um

33:25 So check that. OK. All . Um Back to here.

33:32 So, uh so again, we'll now get into kind of more

33:39 of specific stuff. This is more less kind of an overview which

33:42 you know, again, if you're very comfortable with this,

33:46 you know, then, then, , good, good. That's

33:49 Uh Hopefully, if you're not kind a little bit, not so

33:53 hopefully this has helped. Um But we're gonna kind of get into how

33:57 gene organization in uh So let's start this question here. OK. So

34:06 of these teams are, which of terms includes all of the others?

34:11 . You open a poll there. , um, so it's gonna be

34:16 some terms you're not familiar with because , there's some differences, of course

34:20 how pro Caros organize their genes versus Kaos. So it's gonna be some

34:26 , um, that you may not heard before. So, and there's

34:31 one, a couple in here you not be familiar with. So that's

34:35 gonna go through that in this next here. OK. Let me put

34:54 timer on. OK. Cut All right. Um Yeah,

35:27 There's gonna be genome, right? , uh if we're gonna rank

35:31 it would be a gene from, smallest to largest, let's say um

35:38 being the smallest. No, of not. Nucleotide is small. Excuse

35:43 , nucleotide, then gene, then , then Regulon, then G dot

35:58 . So um that so small is biggest, right? In terms of

36:06 . OK. So, um so we're gonna do is first talk a

36:16 about getting scope. So genome transcript prote genes transcripts proteins. OK.

36:24 for procaryotes, um for us, genome, of course is our

36:31 OK? Also for bacteria and the , but in addition to their to

36:36 chromosome, they may have one or plass. OK. We'll talk about

36:43 and a little bit. Uh you like extra chromosomal small genetic elements.

36:50 . But it does represent part of genome. OK. Uh The transcriptome

36:54 gonna be whatever transcripts are, are that cell at a given time.

37:00 . And that, and a prote fluctuate, right? Because transcript the

37:05 expression, you know, will determine which tran, which transcripts are available

37:10 a given time. And then that course, determines what proteins will be

37:14 from those transcripts. So the transcription can fluctuate and depending on conditions of

37:21 cell of the cell and what it . Um But the genome of course

37:25 , is, is the constant uh terms of genome sizes. So,

37:29 know, on the larger end, pro of GEOM is on the order

37:33 maybe six million base pairs. Uh coli I think is around four

37:39 Um smallest you one of those uh Michaels, those those ones that lack

37:46 cell wall. Uh those are on smaller end like 500,000. So I

37:51 1000 to almost uh um nine almost a billion base pairs. Uh

38:03 million. I'm sorry. Uh you know, within that range,

38:06 , probably about one million is what most mature are typically. Uh But

38:11 they can also have so they're, haploid for the most part, although

38:14 are some, some odd balls that fit that mold. But for the

38:19 part, cars are haploid with 11 and again, may have one or

38:24 of, of these plass. Um All right. So let's

38:29 this is gonna be one of those that we're gonna look at now,

38:35 go over the answer, but then gonna see it again at the

38:37 OK? So it's gonna be one those, I guess before and after

38:40 . OK. So let me So you can read this. So

38:44 is gonna be uh really going through the, the different terminology uh that

38:49 associate with prokaryotes and how they organize genes. So, while reading

39:25 so what we're gonna do is I'm show you a diagram of how you

39:32 out the genes are organized and it's for reference purposes just for comparison.

39:39 not going to test you on your pain structure. OK? But

39:44 it's helpful to see that and then how bacterial proc uh genes differ.

40:12 ? OK. I'm gonna start the . OK. OK. So let's

40:48 here. Well, consensus was f believe. OK, most people pick

40:59 we'll revisit that question a little So let's go ahead. All

41:06 So just briefly before I get e cheese. So just to give you

41:11 little bit of comparison here, um structural genes. So the structural

41:19 um are the ones that code for typically. OK. So we're just

41:24 , we're just gonna worry about protein genes which, which comprise the ball

41:29 the genome anyway. Um So uh , protein coding genes, OK?

41:35 control elements. OK. The uh promoters, uh regulatory sequences, these

41:43 all involved in controlling expression. Then we look at operon and what

41:48 Regulon is. OK. So this Cistron, you see there.

41:52 Cistron, I think it's kind of older term. Um It basically just

41:57 a gene, but you're gonna see like mono systems cynic in the context

42:02 a transcript. OK. So a Syron R N A that transcript contains

42:08 for just one gene. We'll see Caros can form polycystic R N A

42:14 and so that transcript will contain information multiple genes in that single transcript.

42:22 . That's unique to uh pro OK. So let's just first take

42:29 look at you carry out a This is how it, this is

42:33 it is in your, in your . OK. So the first zero

42:39 first on the Exxon intron, So this right here. Well,

42:45 start over here. Control elements enhancer . So all that means is are

42:51 close to the structural genes or far ? OK. The proximal elements are

42:57 course close by enhancer elements can be away, thousands of pairs away.

43:04 . Um Now, what's unique about periodic gene structure? And are some

43:10 IKEA have, have some similarities not all um but the E Exxon

43:18 um uh structure. OK. One to point out is that regardless of

43:25 type of organism you are pro you caros, right? In terms

43:31 gene structure, the constants are a . OK? And regulatory health,

43:39 are gonna be common for any, g you're gonna have a promoter,

43:43 gonna have then a a sequence after promoter that codes for the actual to

43:50 information for a protein, right? the promoters will kind of lines it

43:56 . So remember a, a prelimerase what produces the transcript. But you

44:00 get a alim in front of the and the promoter is what facilitates

44:05 OK. So you're not gonna see gene in anything. It doesn't have

44:09 promoter in front of it. Uh The promoter can also be involved

44:14 regulate breaking for regul regulation as OK? But the promoter is a

44:21 . You see that, see that every gene, right? Promoter then

44:24 the information to code for that OK? All right, back to

44:31 gene. So Exxon Enron, so Exxon sequences are those that actually um

44:37 the information to produce a polypeptide. . The enrons come in between say

44:43 , the, the I N is intervening sequences. So enrons come between

44:47 . OK. So when that's when is transcribed into a, what we

44:54 a primary R N A transcript or M R N A sometimes called,

45:01 contains both exons and enrons. So now we have an R A

45:06 containing both exxons and introns. There also other elements. So you see

45:10 , you see a, a a tail is what it's called,

45:14 call it signal um a five prime , right? These are elements we're

45:20 to see. And you see down . So lots of processing occurs of

45:24 N A in New Caros, And the processing is necessary to take

45:29 the enrons and then what we call together the Exxon sequences. So and

45:36 putting on these modifications and what's called five prime tap and a poly a

45:41 . So that's what we call a M R N A. OK.

45:46 an M R N A that can translated. All right, the pre

45:49 R N A cannot be. So what we have to go through this

45:52 . Um the axons, right can we just label these very simply uh

46:01 and three, these can be spliced in different orders. OK? And

46:08 can create slightly different proteins. So what you cars can do.

46:13 And uh and the cap and tail remember that these, these, this

46:18 you're seeing on the screen. Now is what happens in the nucleus,

46:22 ? So then these M R N S have to exit the nucleus outside

46:27 and then the er and the cytosol become translated. OK. So the

46:32 and tail have a couple of functions help, help it get out of

46:37 nucleus, uh maintain stability of the R N A. Um any M

46:44 N A s that don't have the and tail are rapidly degraded. So

46:49 needs those features to exit the nucleus you remain stable for a period of

46:53 and be translated. OK. So of this do you see in

47:00 OK. So um uh of they have control elements. Uh

47:07 and there's a promoter but there's none this already processing or, or Enron

47:12 structure. OK. So let's flip what it does look like in the

47:18 , right? And, and so , those uh this right here that's

47:27 to designate uh genes that may be continuous, they could be far

47:36 far in the other direction away that's what that means. OK.

47:41 So here you see the single a single promoter, single gene structure

47:47 here, right? one promoter and right. Bacteria and archaea can have

47:58 called an opera structure, right? you have one promoter and multiple structural

48:05 . OK. So promoter and then and more structural genes, two or

48:12 sorry, two or more structural genes are, that typically will be

48:17 OK. And so the only accel of course will bind to the motor

48:24 . And here is where you see Polycystic message. So it's one continuous

48:31 A R A, a transcript that the information for all in this

48:35 all three structural genes on one OK. Very efficient. OK.

48:43 so uh of course, they get into the individual proteins very typically

48:49 right? And so they will be of a common metabolic pathway.

48:56 So this enables bacteria to turn on turn off expression of an entire metabolic

49:03 , which is a very efficient way do it. OK? Not just

49:07 it on but very quickly turn it altogether as well. OK. So

49:12 is a the classic operon structure. . So the Aron itself also includes

49:19 operator. So promoter operator, structural , that's, that's the opera.

49:28 . So um so the operator has function, all right, a little

49:33 different function. It's involved in the . OK. Regulation in conjunction with

49:39 regulatory protein. OK. That's produced a regulatory team. OK. So

49:46 protein very often interacts with the operator . OK. And basically producing a

49:54 block, right? Can't get around . The can't get around to

49:59 OK. So that's a mechanism that's common in pro Caros. OK.

50:07 so um now what we'll see is conditions that bring about this regulatory

50:18 operator interaction to affect transcription, to , to affect expression uh will

50:27 right? We're gonna look at the operon and the Tripen operon and they

50:34 turn off expression bye AAA protein binding operator. But the conditions under which

50:43 happens are very different. So, so there's gonna be variations we'll see

50:48 , on this feed. OK? This is also what we call transcription

50:56 . OK? Because we're affecting the of that opera. OK? Any

51:03 you're interfering with R N A plum its ability to do or not do

51:07 job. That's transcription control. This is a transcription of control mechanism

51:13 common in, in uh pro OK. So, uh so that's

51:22 opera structure. OK. So it, it comprises uh the

51:26 Is this all right here? Our motor operators about two can I

51:35 now the Regulon, OK. So is a single opera. OK.

51:45 , there will be processes uh OK. Uh That involve uh more

51:55 one operator. OK? And when involve more than one operator, you

52:02 can have what's called a Regulon. ? Um A very good example of

52:08 is um controlling multiple operon through a factor. So we'll talk about Sigma

52:16 at the end here uh today, a Sigma factor. So you see

52:21 you have Ayra, OK. A of this is a Sigma factor that

52:27 kind of a transient part because the factor will guide the A pli to

52:31 promoter. And then then once it that the Sigma factor falls off and

52:37 it binds to another a eli right? So Sigma factors guide the

52:41 race to various promoters OK. And you have a number of operon in

52:50 those promoters respond to the same sigma , then you can control those operon

52:58 , right? So here would be example in this diagram that this Sigma

53:06 is common to these promoters in these OK. So that's how you can

53:17 control of these various operon. And so why would you do

53:23 Well, because presumably the, the are all a part of a particular

53:31 ? Ok. A good example is , what we call the nitrogen

53:36 Ok. So think of all the pathways in which nitrogen might be

53:44 right? We need nitrogen to form acid nitrogen to produce nucleotides. We

53:50 through um previously a lecture uh about , you know, and it's used

53:57 either in aerobic expiration or as a AAA food source for a little,

54:05 ? So all it represent can different in the same cell. OK.

54:11 nitrogen coming in then will be, know, controlled in terms of where

54:16 goes this pathway and that pathway and other. And for that reason,

54:21 kind of want to control the OPERON that are all involved in these different

54:27 of nitrogen metabolism. OK? That's example. OK. Um And that's

54:33 you do it, you control the together to kind of make sure that

54:37 nitrogen coming in is allocated as it be for, for most efficient

54:43 OK? Um And so that's what Regulon is. It's a way to

54:48 multiple OPERON that are part of a part of a common metabolism,

54:53 That nitrogen be right. Um We'll see it in chapter in chapter

54:59 we see a Regulon that's involved in in grand positive organisms. And uh

55:07 again, because it's, it's using number of OPERON to, to bring

55:12 this, this uh transformation bringing in from the environment. So again,

55:18 are you at? Where, where it's a process involving multiple

55:22 Um You can control those various OPERON a Regulon. And again, it's

55:29 that Sigma factor kind of being common those motors of that particular of that

55:34 Regulon and controlling those opera together. . So that's, that's basically an

55:39 . So a Regulon is AAA control that controls multiple opera at the same

55:50 . OK. Uh Let me just over here. Any questions.

55:59 Um Got it again, feel free chime in on a problem wondering.

56:10 OK. So let's look at this on plasmids. So we're going to

56:16 about plasmids for a second or a minutes. Um So, um so

56:26 are uh we finished today, so talk about OPERON and um then

56:35 we're gonna revisit that again when we to chapter 10 on um regulation,

56:44 ? So, in regulation, of , we're looking at opera and how

56:47 controlled in various ways we look at couple of different examples. Um When

56:53 get, when we get to next in chapter nine, that's more about

56:58 horizontal gene transfer, how bacteria and can uh transfer genes between them.

57:08 . That's separate from, from um through, through replication, right?

57:17 binary fission, a cell splits in , those two daughter cells receive that

57:23 , right? That's one mode of . But there's also another mode where

57:26 can exchange DNA genes with members of population. And that's what like

57:33 transformation transduction transposition is all about. . We'll look at that next

57:40 Um But plasmids are a, are part of that as well.

57:45 So that's, we'll see that you , you can um move, move

57:50 around uh through classmates. OK. let me go ahead and set the

58:35 . OK. You know, OK. Let's see. Um

58:50 Uh C C is the true is correct statement here. OK. Um

58:56 plasmas have this feature of being what call a OK? They don't um

59:02 kind of to a certain extent, things on their own. They're not

59:06 to um replication of the chromosomes. remember the chromosome in a cell replicates

59:13 to cell division, that's kind of it's linked to uh plasmas can kind

59:16 do the wrong thing in that Uh They're not always retained. Uh

59:23 on to a plasmid is, is really dependent on uh is there a

59:32 for the cell to hold on to ? And there's factors that play into

59:35 as we'll talk about. OK. they do have a, they can

59:39 a different way of replicating uh from , for example, our chromosome

59:45 Um So let's talk a little bit plasmids. So again, these are

59:51 chromosome elements. So you see this a, an E coli, I

59:54 that's been gently lic. So you the larger chromosome spilling out right threads

60:02 I've circled the what the actual, actual it's like right here.

60:11 Um Right here. OK. So see how small it is compared to

60:20 larger chromosome. And again, an size of a plasma is typically around

60:26 to 10,000 bases. OK. There be some that are on the larger

60:31 upwards to 50 to 100,000. But the more typical ones are much less

60:36 that. Um They um because they so the elements that make them

60:44 right, the or sequence, This is what this is where uh

60:50 is where replication initiates, right? um the uh OK. So the

61:06 because it has its own orbital it can replicate independent of the larger

61:11 . OK. And this plasmin So plasmas of course originated in with

61:17 . But when they were discovered, are 40 plus years ago by

61:23 um we've taken these plasmas out of and have engineered them for our own

61:30 . So if you may or may know plasmas are a vectors,

61:35 Or plasmas. These are uh really in um in the recoin DNA,

61:42 ? In, in, in uh them to carry various genes and

61:46 So we, we construct them for own purposes. So uh the,

61:52 other terms you see here like the three bam H one um pst

62:00 these are restriction enzyme sites. So can cut plasmids with restriction enzymes.

62:05 then, and then we combine in other genetic elements. That's how you

62:10 manipulate a plasmid and, and, , and construct it for your own

62:14 . OK. And so in this , we see uh a couple of

62:18 , the AMP which is Apollon tetracycline, which is for tetracycline

62:25 So, plasmas um because they're a size, they can kind of be

62:30 transferred between cells by different methods, by a conjugation. And in doing

62:37 , whatever plasmas are transferred, the cell will receive a course of plasma

62:43 the ability to express whatever genes are that plasma. OK. So um

62:49 number that also relates to or and can have, you can actually have

62:55 or in a plasma and one may may be for when it, when

63:00 um conjugating, one may be what , they often will dictate whether

63:05 whether it's high or low. And simply means how many copies are produced

63:09 cell. OK. So high copy can be anything from upwards of around

63:14 per cell. Low copy number OK. And so plasma is varied

63:20 that's typically dictated by the that they , whether it's a high or low

63:25 number. OK. Um We will next week when we talk more about

63:32 and conjugation how they could be transferred the cell to cell that they could

63:37 into the chromosome. So that, is true. OK. And

63:41 of course, by integrating to the that can kind of make them a

63:46 permanent resident of the cell. Uh But they can also, if

63:53 integrate, they can also actually come and exist as a extra chromosomal plasma

63:58 well. So they can go both . OK. Um And transferable.

64:03 are transferrable and so you can transfer cells of the same species, sometimes

64:09 different species. OK. And so we can classify plasmids in different

64:15 uh these are a couple of ways factor F factor cata plasmids based on

64:21 kind of genes they're carrying. So this one, since it has

64:27 resistance and hyper resistance, you might this A an R factor,

64:31 It has genes for antibiotic resistance. an F factor is what makes it

64:37 we call mobile, makes it OK. And so um this could

64:43 this pattern itself could be that way um let me just get this out

64:49 the way here if we um if were to like uh have a,

65:00 a jean in here, right, we call the factor, OK.

65:11 that factor means it has the sets genes needed to make it transferable and

65:16 components that, that are part of process. And so if it has

65:21 , we can say it has the factor and so what can be transferred

65:25 whatever other genes are in there? right. So for example, a

65:30 and tempo resistance. So the fact now we have an F factor in

65:34 , it makes it transferable and those resistances can be passed on as

65:38 OK. Kind of al simply just a 345 genes involved in a particular

65:46 pathway. Very common is like right? We talked about aromatic uh

65:51 of aromatic compounds. So those those often small pathways that can be found

65:55 plasmin. And again, if it that factor, it can be transferable

65:59 other cells. OK. Um So replication. So bidirectional replication is

66:08 you're familiar with. That's what you . Um you know, separate the

66:13 of the right. And then you , you create the two forks,

66:17 ? And then you carry out bidirectional , right, um forming two

66:24 What we saw before now can also by a mechanism called rolling circle

66:32 OK. So how that begins is the formation of a of a niche

66:37 which is basically a a cleavage of covalent bond right in the sugar prostrate

66:48 bone. OK. And in doing , you expose the three prime hydroxyl

66:55 of a nucleotide. So if you , you know kind of deification,

66:59 know that DNA polymerase works by adding to the three prime hydroxyl end,

67:07 ? That's how you synthesize DNA. if you expose that three prime

67:10 the DNA Climara can then begin to from that. Of course, what

67:14 copying, it's copying the template Of the of the complementary strand as

67:19 doing that. OK. And so happens is, so here's what

67:24 we had create Nick, right? is the rep a replicates a enzyme

67:29 the one that does this. So create a Nick, right? So

67:32 expose that three prime in and from , you can um the plym can

67:40 , right? And of course, you, it's copying uh this is

67:44 template, it's cocky, right? so as it does, it's displacing

67:52 strand. So you see how this here is being displaced, right?

67:57 we we're synthesizing new DNA from that , right? So that's being displaced

68:03 as it continues, right? So see the dark purple is the new

68:07 for creating a copy. Now, this other strand that's been displaced

68:13 right? It too can be right? So you have um uh

68:18 and then you do the whole copying of that template and uh you create

68:24 double stranded copy of that. All . And so that's what we see

68:29 conjugation. So, so imagine if had some in conjugation, two cells

68:36 coming together, so it could be cell, right? And this would

68:41 the other. So OK. And we'll see this process next week,

68:47 ? So in one cell as a , every as we're doing a growing

68:52 replication, the other strand as is displaced is shoveled into a a cell

69:00 mating with. OK. And so this other cell will get a copy

69:06 that. Here's the one cell, the other cell. And so in

69:10 other cell, it'll make the complementary of that and it too will have

69:14 copy of that plasmin. OK. that's how you can transfer plasma plasmas

69:19 cells through components that bring bring about connection between the cells. And then

69:25 circle replication enables the the recipient cell get a copy of that plasmin.

69:31 again, the replication we'll see again the context of conjugation where we're transferring

69:37 plasmin text. So as mentioned earlier that question, you know, we

69:44 the cells that rep painting, retaining um um plasmin itself, right?

69:54 And so what are the factors involved that? And so selective pressure,

70:00 is what it's about low copy number high copy number is also a

70:05 OK. So think of a cell divided by binary fission. OK?

70:11 if you're a low copy number or sorry, a high copy number of

70:15 in that cell and that cell begins divide. Well, no matter if

70:21 , you know, divides that plane however, right, because there's so

70:27 plasmas, you know that at least is gonna end up in the daughter

70:32 , right? So um so for copy number plasmids, you know,

70:37 not, it can be an But you know, initially it's,

70:40 it's it's the likelihood is very high both the daughter cells will receive a

70:45 of the plasmin. But what if low copy number, right? If

70:48 a low copy number plasmin, you can't just rely on the fact

70:53 you know, division will occur such each gets a copy, right?

70:57 So to kind of improve the odds cells have what are called these P

71:03 R or par proteins that's short for . OK. And so this,

71:08 kind of like a quasi pseudo mitotic if you will, if you recall

71:13 from mitosis, uh it's not but it kind of looks something similar

71:17 of action as that. So the proteins actually bind to the plasmid as

71:23 see there, then they polymerize and as they polymerize, they kind of

71:29 um um push each plasmid to opposite of the cell. OK. And

71:36 then when the cell divides, then know, each daughter cell receives a

71:40 of the plasmin. So that's, kind of a unique situation for those

71:44 have these like one, maybe one two copies in the cell.

71:48 And so having this mechanism kind of that, that, that, that

71:53 the cell divides each cell gets a of that plasmin, OK. But

71:58 know, back to selective pressure. even if you're a high copy number

72:02 plasmin, you know, the the cell can actually lose those over

72:06 if you don't have selective pressure on . OK. So what does that

72:11 ? Well, that just shows you of paid uh what what this,

72:15 happens as the part proteins polymerize the go to poles and the cell would

72:22 , right? And then each cell a copy of that. So back

72:26 selective pressure. OK. So here's basic example, right? So here's

72:32 E coli and it's carrying a plastic tetracycline resistance. OK? There's an

72:38 coli that doesn't have that plastic. it's sensitive, it's, it's uh

72:43 . OK. So, so if we're gonna uh the E coli

72:48 tetracycline sensitivity will not grow on the not grow on this medium with,

72:55 tetracycline, right? It's gonna be to it, the tetra resistant one

73:00 course, can OK. So will ecoli grow on both the types?

73:06 course, because, you know, though it's resistance, right?

73:11 it's resistance. All right. That's , that's, that's what the superscript

73:15 means. So it will grow on to, on uh on uh tetracycline

73:22 medium with tetracycline because it's resistant to . Ok. The um but it

73:29 grew on here as well, of , because it's, it doesn't matter

73:35 it's has a resistance, there's no in the media. So it'll grow

73:38 as well. Now, the thing OK. If you, if you

73:46 to grow it on this medium OK. If you continue to grow

73:51 on that and then every so every every couple of weeks or so,

73:57 then transfer to fresh media and you doing that over and over,

74:01 You can go from here uh to media, right? Um That will

74:10 eventually without tetracycline being present, The cells will lose that plasmid,

74:19 ? Because there's no selective pressure to it right. So again, this

74:23 back to the same thing we keep about is deficiency and energy,

74:30 It takes energy to maintain that OK. Why? Well, you

74:34 to replicate it. All right, go through cell division and you have

74:37 replicate the plasmid, that's extra energy . OK? If there's no pressure

74:44 keep that plasma. In other if there's no, if, if

74:47 doesn't provide that cell with a selective , then why hold on to it

74:52 after several, you know, passages medium, it will lose that

74:57 Ok. That's why if you if you have any cholera and you

75:01 a plasmid with a certain gene in , you want, and you want

75:04 keep, want to hold on to . Well, then you put,

75:08 put an antibiotic, resistance gene in and you keep it on medium with

75:13 antibiotic that will ensure that that cell hold along that plasmid. OK?

75:20 it's, it's for its own survival do so, right? So the

75:25 there can, so certainly things that , that are on plan genes on

75:29 are not always critical for survival for , for the cell that has

75:32 but it can be in some So if it does have it,

75:36 that will cells that possess that plast of course be the ones that survive

75:40 they'll pass that plast on to others this population. Ok? But if

75:45 selective pressure is not there and and it continues to not be

75:50 then there is a danger that the will be lost, right? Because

75:54 , it becomes, it boil down uh efficiency and energy, right?

76:00 You know, is it worth the energy expenditure to hold on to this

76:03 if I'm not eating it? So that's kind of what's going on

76:08 ? Ok. Um Any questions about ? Ok. So, um,

76:21 let, let me um I'll tell what I know we're almost running out

76:25 time. But let me uh I'll go through this a little bit about

76:29 a plume race and then we'll wrap continue with the can on Monday.

76:36 , OK, so that, so chapter eight, this is the only

76:39 I'm really talking about here is, a and a little bit about

76:44 OK. So with bacteria, Ara there's a part of course that

76:51 will um um synthesize the R N from the DNA template, right?

76:58 the, the core R A plume has these four subjects. And this

77:02 what brings about the synthesis of the N A. OK. The sigma

77:06 is more transient. In other it will, it certainly will

77:10 right? So it's, it's right , this little red blob, it's

77:14 Sigma factor. And so it's what it to the promoter. OK.

77:19 so by binding uh to the it puts it in front of the

77:24 . And so it kind of acts scanning and looking for these what I

77:29 minus 35 minus 10 region. This is a region that's very common

77:35 pro promoters. OK. So Sigma looks for that and while it's attached

77:43 the ali, it brings it to area and once it does, so

77:50 strand separation occurs and you begin to the gene. OK? And so

77:57 promoter is gonna be set up right front of that gene. So as

77:59 begins copying, it's gonna copy that coated sequence. OK? And so

78:07 that begins to happen, then the sigma factor will fall off.

78:12 . But then it can, is to bind another R N A

78:14 bring it to the promoter and, , and initiate the process again.

78:19 . That's what Sigma factor does. that's how, that's, you'll see

78:23 factors in, in you car your ac it works differently. But for

78:28 and art heal polymerase, that's how works with a Sigma factor that guides

78:33 to the pro boder. OK. so the, the minus 35 minus

78:38 sequences. OK. Uh These are we call consensus. In other

78:43 when we looked at numerous but uh from motors, we always see very

78:49 sequences in these two regions. And the minus 35 minus 10 it refers

78:54 upstream from the start side of OK. So either 10 nus upstream

79:02 the 35 upstream from the start in two regions, we see very similar

79:08 . OK. So here it gives example. So Sigma 70 is a

79:13 common sigma factor. OK? And think the 70 relates to how big

79:18 is the mask. Uh So it's very big, well, it's,

79:23 , it's one that's been found in lot of genes in, in uh

79:27 . And so, um so you here in yellow or both minus 35

79:35 10, the uh consensus sequences. they're very common among all these different

79:42 promoters of E COLI, for OK. And you can stretch that

79:46 different different bacterial species. They have in, in these, in these

79:51 35 minus 10 regions to see this commonality in the OK. That's a

79:58 consensus sequence. So here you see start of transcription here right there plus

80:06 . OK. So use minus 10 minus 25 upstream from that. And

80:11 you know, we talk about promoter that really uh is about the level

80:17 expression. That's what that correlates OK is um levels of expression.

80:24 strong promoter will have high levels of compared to a weak promoter.

80:30 And you can manipulate these things, can. So that's what's done oftentimes

80:34 in biotechnology, when you want if you have a particular protein that

80:39 , that a bacteria makes and you want to enhance production of that

80:44 In other words, enhance expression. oftentimes begin by manipulating the factors that

80:50 expression. And a promoter sequence is of those. OK. So you

80:54 , you can alter the, the in within the sequence to maybe increase

81:00 in some cases, maybe it lowers . OK. So that's what down

81:03 up. So an up an up uh change is what increases activity

81:10 one decreases and so that, that bring that bring that about. And

81:16 um the uh so the minus um again, it is very common,

81:22 ? Most genes that's the Sigma factor controls it. OK? And or

81:28 uh Sigma 70 factor is what binds those promoters and it shapes expression.

81:34 ? Um So the minus 25 so minus 25 minus 10 is, is

81:38 common thing also not just for the 70 but also for the uh 32

81:45 28 right? These also have that of sequences a little bit different.

81:51 But these other Sigma factors are involved different functions. So things like heat

81:56 , we'll talk about that later. heat motility, uh stress response.

82:01 these have their own particular Sigma factors their particular sequences they respond to and

82:07 a different one here. This is we looked at earlier in the context

82:10 Regulon, right? The N 54 sigma 54 is nitrogen metabolism,

82:17 So that nitrogen Regulon, right is because the OPERON for that, for

82:23 particular Regulon all respond to that particular factor. So that's how you can

82:27 of control those coordinately, right? So uh that's a, that's a

82:34 think a good place to stop. We'll do some of this limited review

82:40 um uh next week. Uh So next Tuesday we're gonna meet in class

82:48 hopefully for the rest of the it will be that way.

82:51 And so, um, and if you, uh, if you

82:58 have any questions, um, then will see you all in person next

83:04 and, uh, we already has of the stuff at the beginning of

83:07 next time. Um Anyway, if no questions, folks, uh we

83:13 see you next week in person. . And, uh, I will

83:21 this recording and so you can review this afternoon or whenever, uh,

83:26 you like. Ok, thanks,

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